Integrated Responses of a Wild Species Parietaria Judaica (L. 1753) to Direct and Induced Iron Deficiency Growth Conditions

Ph.D. GRADUATE SCHOOL IN Land, Environment and Biodiversity

DEPARTMENT OF Agricultural and Environmental Science – Production, Landscape and Agroenergy

COURSE IN Agricultural Ecology – cycle XXV

Integrated responses of a wild species Parietaria judaica (L. 1753) to direct and induced iron deficiency growth conditions

DISCIPLINARY SCIENTIFIC FIELD - AGR/13 -

DOCTORAL CANDIDATE Liliana Maria Tato DOCTORAL ADVISOR Prof. Graziano Zocchi

Ph.D. SCHOOL COORDINATOR Prof. Graziano Zocchi

A.A. 2011-2012 i

Activities carried out during the three-year PhD

Common Courses of the Ph.D. Graduate School Land, Environment and Biodiversity

- Technical Imaging Analysis (Obligatory Course) - Stella Pratissoli Università degli studi di Milano January - February 2010 - Instrument Analysis (Obligatory Course) Università degli studi di Milano March - April 2010 - Scientific communication (Obligatory Course) - Davide Vignati Università degli studi di MilanoMarch - April 2010 - Reactive Transport in porous media: applications to contaminant hydrology bioremediation, colloid transport, and groundwater age dating - Prof. Timothy R. Ginn – University of California at Davis – USA Università degli studi di Milano October 2010

International PhD School

- International PhD Summer school Innovative Strategies to Improve Winemaking: new perspectives in the light of new knowledge Università degli Studi di Milano - Di.Pro.Ve, Bolla Cellar - Pedemonte – VR July 2011 - International PhD summer school, Soil, plant and biomasses in a changing environment Società Italiana di Chimica Agraria, Pieve Tesino (TN) May 2012

Scientific experiences in National Research Institutions

- Istituto di Scienze delle produzioni Alimentari (ISPA) - Exudates Analysis by HPLC/ Mass CNR Bari - July 2012 - Dipartimento di Biotecnologie per il Monitoraggio Agro-alimentare ed Ambientale - Imaging Analysis of roots (Winrhizo) - Università degli Studi Mediterranea - Reggio Calabria October 2012 ii

National Congresses

- XXIX Convegno Nazionale - Società Italiana di Chimica Agraria Università degli Studi di Foggia – Foggia 21-23 September 2011

International Congresses

- FISV 2012 XII Congress Università La Sapienza – Roma, 24-27 September 2012 - ICP 2012 - XXVI International Conference on Polyphenols Università di Firenze, 22-26 July 2012

International Publications (ISI)

- L. Tato, P. Tremolada, B. Cristiano, N. Guazzoni, M. Parolini, M. Caccianiga, A. Binelli, 2011. Seasonal and spatial variability of polychorobiphenyls (PCBs) in vegetation and cow milk from a high altitude pasture in the Italian Alps, Environmental Pollution, - S. Donnini, P. De Nisi, D. Gabotti, L. Tato, G. Zocchi, 2012. Adaptive strategies of Parietaria diffusa (M.&K.) to calcareous habitat with limited iron availability, Plant, Cell & Environment, 35:6, 1171-1184 - L. Tato, P. De Nisi, S. Donnini, G. Zocchi, 2012. Low iron availability influences phenolic metabolism in a wild plant species (Parietaria judaica L.), PPB special issue, submitted

National and International poster presentation

- P. Tremolada, N. Guazzoni, L. Tato. A model for the evaluation of vegetation to milk transfer of Persistent Organic Pollutants (POPs): the Cow model, XX Congresso SITE 2010 - "Sapienza" Università, Roma 27-30 September 2010 iii

- L. Tato, P. De Nisi, S. Donnini, G. Zocchi. Adeguamento metabolico in Parietaria judaica L., in condizioni di bassa biodisponibilità di ferro, XXX Convegno Nazionale SICA 2012 – Milano 18-19 September 2012 - L. Tato, P. De Nisi, S. Donnini, G. Zocchi Low iron availability and phenolic metabolism in a wild plant species (Parietaria judaica, L.), XXVIth International Conference on Polyphenols - Università di Firenze – Firenze 22-26 July 2012

National and international oral presentation

- L.Tato, G. Zocchi. Risposte adattative di Parietaria judaica sottoposta a ridotta disponibilità di ferro indotta da diverse condizioni di allevamento, XXIX Convegno Nazionale - Società Italiana di Chimica Agraria - Università degli Studi di Foggia – Foggia 21-23 September 2011 - L. Tato, P. De Nisi, S. Donnini, G. Zocchi. Integrated responses of Parietaria judaica to iron deficiency conditions, 12th FISV Congress - Università di Roma La Sapienza – Roma 24-27 September 2012

Seminar presentation on progress research activity during PHD

- Study of adaptative responses to calcareous substrate and alkaline pH in wild species (Parietaria judaica) - Integrated responses of Parietaria judaica to iron deficiency conditions

Participation to Seminars

- La Twilight Zone del Mediterraneo: meraviglie nascoste a portata di ROV - Prof. Marzia Bo - Università di Ancona Università degli studi di Milano 13-gen-10 - Cetacei in mar Mediterraneo - Dott. Marco Colla Università degli studi di Milano 20-gen-10 iv

- Mineral Fertilization of Fruit Trees: Basic knowledge and dissemination - Prof. Volker Roemheld University Hohenheim – Stuttgart DI.PRO.VE - Università degli studi di Milano 06-set-10 - Utilisation of Halophyte species for saline soil rehabilitation and valorisation - Prof. Chedly Abdelly - Laboratoire des plantes Extremophiles - Centre de Biotechnologie de Borj Cedria – Tunisia DI.PRO.VE - Università degli studi di Milano 25-nov-10 - Strategies of salt tolerance in halophites - Prof. Chedly Abdelly - Laboratoire des plantes Extremophiles - Centre de Biotechnologie de Borj Cedria – Tunisia DI.PRO.VE - Università degli studi di Milano 25- nov-10 - La fluorescenza della clorofilla come metodologia di studio degli stress nelle piante - Dott.ssa Lucia Guidi - Dip.Biologia delle Piante Agrarie - Università di Pisa DI.PRO.VE - Università degli studi di Milano 27-gen-11 - Analisi Statistica dei Dati con SPSS - Dott.ssa Paola Aguglia - SPSS Italia Seminario tenuto allo StarHotel Ritz – Milano 23-mar-11 - Strumenti e tecniche di misura per il bilancio di acqua e carbonio negli agroecosistemi - Dip. Ingegneria Agraria Workshop tenutosi a Landriano (PV) 05-ott-11

Teaching activity

- 2010, October (4 hours): General Biology Course -Agricultural Faculty - Università degli Studi di Milano - 2011, January-February (13 hours): practice lessons for Botany Course (bachelor degree in ‘Scienze Viticole ed Enologia’) Università degli Studi di Milano - 2011, October (4 hours): General Biology Course - Agricultural Faculty - Università degli Studi di Milano - 2012, March (8 hours): practice lessons for Biology Course (bachelor degree in in ‘Scienze Viticole ed Enologia’) Università degli Studi di Milano - 2012 October (4 hours): General Biology Course -Agricultural Faculty - Università degli Studi di Milano v

M.Sc. Liliana M. Tato attended the three year PhD studies with diligence, perseverance and responsibility. She has shown great interest in the topics addressed demonstrating ability in critical analysis towards the issues faced during the research activities. She has acquired a very good knowledge of the eco physiological basis inherent the issue studied as well as the physiological and biochemical techniques for their study. She has independently set collaborations with other research groups in order to investigate specific aspects of the research, thus acquiring new analytical techniques. She attended for a short period the Instituto di Scienze delle Produzioni Alimentari (ISPA), Consiglio Nazionale delle Ricerche (CNR), Bari – Italy under the supervision of Prof. Vincenzo Lattanzio applying the HPLC technique to the study of phenolic compounds; and the Dipartimento di Agraria – Università Mediterranea di Reggio Calabria under the supervision of Prof. Maria Rosa Abenavoli, where she acquired the root image analysis technique (winrhizo) to study the modification of root morphology and architecture. Therefore I express an excellent opinion regarding the work carried out by Liliana Tato. vi

to my mother who taught me the art of never giving up

to Fernando, Ramiro, Clara and Gregorio who supported and put up with me in this adventure

vii

ABSTRACT

Iron (Fe) is an essential micronutrient in plants as it takes part in major metabolic pathways such as photosynthesis and respiration and is linked to many enzymes that accomplish many other cellular functions (DNA synthesis, nitrogen fixation, hormone production). Fe deficiency, a major abiotic stress, reduces crop yields, especially in calcareous soils in which the solubility of Fe is extremely low because of the high soil pH. Parietaria judaica is a wild sinantropic Strategy I plant that implements many integrated mechanisms allowing to successfully complete the life cycle in highly calcareous environments. In this work the main mechanisms by which P. judaica overcomes the low bioavailability of Fe have been identified. P. judaica was subject to direct and induced Fe deficiency growth conditions in hydroponic systems. Strategy I biochemical mechanisms of FeII-reduction and rhizosphere acidification were studied, as well as low organic acids and phenolics in root exudates. It was suggested that the accumulation and exudation of phenolic compounds plays a central role in the adaptive strategy of P. judaica to cope with Fe deficiency conditions. Key enzymes of primary and secondary metabolism were assayed in order to identify the metabolic rearrangement that occurs under Fe deficiency conditions. The data analysed confirm that under Fe deficiency the metabolic rearrangement takes place by modifying allocation of carbon skeletons between primary and secondary metabolism. It was observed that secondary metabolism constitutes the main concern under this stress condition as P. judaica sustains the supply of substrates using non oxidative ways. Phenolic composition was characterized by HPLC analyses in P. judaica focusing on phenolic composition changes due to low Fe availability. Phenolic compounds found in P. judaica belong mainly to the mono- and dicaffeoylchinic acids group. The chlorogenic acid was resulted the most sensible component under Fe deficiency stress. Morphological and architectural modifications of root system were also analysed. P. judaica changes its root system according to the experimental treatment imposed. The differences found in direct and induced Fe-Deficiency conditions were oriented to increase the root contact surface with the medium. In particular, the root architecture reflected the plant nutritional status. viii

Comparing the data obtained from high bicarbonate and highly alkaline buffer conditions it was observed that P. judaica has no problems to acquire Fe in highly alkaline environments, suggesting that in a highly chalky environment the availability of bicarbonate itself constitutes the real factor of stress. ix

AKNOWLEDGMENTS

My first and sincere thanks go to my advisor and mentor, Prof. Graziano Zocchi for the confidence placed in me in accepting my candidacy at the doctoral school. His patience, encouragement, understanding and continuous support made this work successful. I will always appreciate the freedom he gave me to carry out the research, his open mind, the stimulating working environment and the opportunities that he provided me. I extend genuine thanks to Prof. Vincenzo Lattanzio for his continuous availability and for giving me the opportunity to attend the ISPA laboratory. I have greatly benefited from his deep knowledge of all aspects of phenolic compounds as well as his stimulating insights and methodological advice. I also extended my gratitude to Prof. Maria Rosa Abenavoli who generously welcomed me to the Università Mediterranea and gave me all the necessary support to acquire a new technique. I thank her for the considerable time dedicated to this work and for her sincere friendship. I wish to thank Dr. Patrizia De Nisi for her selfless support, advice, for the good time we passed during the Congress in Firenze, the laughs and her great sense of irony. I am deeply indebted to Dr. Marta Dell’Orto for her inestimable help in the critical review of this thesis and other work I have done during the PhD. She always provided me with valuable and stimulating feedback. Thanks also for the good aperitifs. I would not have been able to complete this journey without the aid and support of many people over the past three years: Dr. Silvia Donnini and Dr. Gianpiero Vigani for their availability, suggestions and friendship; Dr. Alessandro Abruzzese and Giorgio Lucchini for their continuous technical support. Finally, I would like to thank very much my family, Fernando, Ramiro, Clara e Gregorio for their endless support, patience, encouragement, and for leaving aside any home cooking demands, without them this experience would not have been possible.

“[…] porque todos son sentencias sacadas de la mesma experiencia, que es la madre de las ciencias todas [….]”

Miguel de Cervantes Saavedra, 1605 (from El Ingenioso Hidalgo don Quijote de la Mancha I, Chapter XXI)

“The most beautiful think we can experience is the mysterious. It is the source of all true art and all science. […]”

Albert Einstein, 1931 (from The world as I see It, Forum and Century, 84:193-194)

TABLE OF CONTENTS

ABSTRACT ...... vii

CHAPTER 1. General Background: Ecophysiology of iron plant nutrition ...... 1

1.1. Iron in the soil ...... 2

1.2. Iron in plant physiology ...... 6

1.3. Iron acquisition in dycotiledoneous plants: the Strategy I ...... 9 1.3.1. Reduction of Fe(II) chelates: a pre-condition to Fe acquisition ...... 10 1.3.2. Fe(II) transport inside the root cell ...... 12 + 1.3.3. Acidification of the rhisosphere; the plroton pump H -ATPase ...... 12 1.3.4. The role of exudates in Fe acquisition ...... 17 1.3.5. Morphological changes induced by Fe deficiency ...... 19

1.4. The iron deficiency in crops ...... 20 1.5. Wild plants strategies to low nutritional deficiencies: the case of iron deficiency ...... 22 1.6. Parietaria judaica: a wild species tolerant to high calcareous Environments ...... 24

1.7. Aim of the research ...... 26

References ...... 28

CHAPTER 2. Adaptive strategies of Parietaria diffusa (M. & K.) to calcareous habitat with limited iron availability ...... 36

2.1. Introduction ...... 36

2.2. Materials and methods ...... 40 2.2.1. Plant Material ...... 40 2.2.1.1. FIELD SAMPLING ...... 40 2.2.1.2. HYDROPONIC CULTURE ...... 40 2.2.2. Collection of root exudates ...... 41 2.2.3. Determination of phenolic compounds ...... 41 2.2.4. Organic acids assay ...... 41 2.2.5. Biomass measurement and leaf chlorophyll determination ...... 42 2.2.6. Determination of Fe and P ...... 42 2.2.7. Fe reduction and acidification in vivo ...... 42 2.2.8. Fe reduction by phenolics exuded and accumulated in the roots ...... 43 2.2.9. Preparation of plasma membrane vesicles ...... 43 + 2.2.10.H -ATPase assay ...... 44 xii

2.2.11.FC-R assay ...... 44 2.2.12.Soluble protein extraction and PEPC assay ...... 44 + 2.2.13.Western blot analysis of PEPC and H -ATPase ...... 45 2.2.34.Shikimate pathway enzyme extraction and assay ...... 45 2.2.15.Protein determination ...... 46 2.2.16.Oxygen consumption ...... 46 2.2.17.Statistics ...... 47

2.3. Results ...... 47 2.3.1. Organic compounds in roots and exudates ...... 47 2.3.2. Biomass measurement ...... 48 2.3.3. Leaf chlorophyll determination ...... 49 2.3.4. Fe and P determination ...... 49 2.3.5. Root Fe reduction and medium acidification in vivo ...... 50 2.3.6. Fe reduction ability by phenolics ...... 51 2.3.7. Strategy I response activities in plasma membrane preparations and root soluble extracts ...... 52 2.3.8. Shikimate pathway enzymes ...... 54 2.3.9. Oxygen consumption ...... 56

2.4. Discussion ...... 57

References ...... 63

CHAPTER 3. Comparison of different alkaline pH growth conditions (bicarbonate and organic buffer media) on Parietaria judaica in a time course analysis ...... 70

3.1. Introduction ...... 71

3.2. Materials and methods ...... 74 3.2.1. Plant material ...... 74 3.2.2. Collection of root exudates ...... 75 3.2.3. Enzymatic determination of organic acids ...... 75 3.2.4. Determination of phenolic content ...... 76 3.2.5. Determination of apoplastic Fe ...... 76 3.2.6. Soluble enzyme extraction and assay of PEPCase and G6PDH ...... 77 3.2.7. Statistical analysis...... 77

3.3. Results ...... 78 3.3.1. Malic and citric acid content in root tissues and exudates ...... 78 3.3.2. Phenolic compounds in root extracts and exudates ...... 81 3.3.3. Root apoplastic Fe...... 83 3.3.4. PEPCase and G6PDH activity ...... 84 xiii

3.4. Discussion ...... 85

References ...... 93 CHAPTER 4. Low iron availability and phenolic metabolism in a wild plant species (Parietaria judaica L.) ...... 98

4.1. Introduction ...... 98

4.2. Results ...... 101 4.2.1. Root modifications ...... 101 4.2.2. Phenolic compounds in root and exudates ...... 102 4.2.3. Responses of primary metabolism ...... 103 4.2.4. Shikimic pathway enzymes ...... 105 4.2.5. Oxidative and non-oxidative pentose phosphate pathway activation (OPPP and non-OPPP) ...... 108

4.3. Discussion ...... 109

4.4. Materials and Methods ...... 115 4.4.1. Plant material ...... 115 4.4.2. Collection of root exudates ...... 115 4.4.3. Determination of phenolic content ...... 116 4.4.4. Soluble enzyme extraction and assay ...... 116 4.4.5. Shikimate pathway enzyme extraction and assay ...... 117 4.4.6. Transketolase enzyme extraction and assay ...... 117 4.4.7. Protein determination ...... 118 4.4.8. Polyacrylamide gel electrophoresis and Western blot analysis of enzymes ...... 118 4.4.9. Statistical Analysis ...... 118

References ...... 119

CHAPTER 5. Characterization of polyphenols in tissues and exudates of Parietaria judaica undergoing direct and induced iron deficiency growth conditions ...... 126

5.1. Introduction ...... 126

5.2. Materials and Methods ...... 129 5.2.1. Parietaria judaica culture ...... 129 5.2.2. Collection of root exudates ...... 130 5.2.3. Isolation and characterization of phenolic compounds in Parietaria judaica ...... 130 5.2.4. Determination of total phenolic compounds ...... 131

xiv

5.2.5. Estimation of total ortho-dihydroxy phenolic compounds content (Arnow’s reagent) ...... 131

5.3. Results and Discussion ...... 132 5.3.1. Phenolic characterization of Parietaria judaica collected from the field ...... 132 5.3.2. Phenolic compounds in roots from different hydroponic treatments (+Fe, -Fe, Bic and Tric) ...... 135

5.4. Conclusions ...... 142

References ...... 143

CHAPTER 6. Morphological and architectural modifications of the root system of Parietaria judaica in response to different Fe deficiency conditions ...... 146

6.1. Introduction ...... 146 6.2. Materials and Methods ...... 148 6.2.1. Plant material and growth conditions ...... 148 6.2.2. Root morphology and biomass allocation ...... 148 6.2.3. Statistical analysis of data ...... 149

6.3. Results and Discussion ...... 150

6.4. Conclusions ...... 160

References ...... 162

CHAPTER 7. Generale conclusions – Summary of findings and future research directions - ...... 166

CHAPTER 1 ______

General background: Ecophysiology of plant iron nutrition. 1

Iron (Fe) is an essential element that participates in major biological processes both in plants and animals. Due to their reversible redox characteristic Fe ions were readily incorporated for use in biological processes early in evolution playing a key role in metabolic pathways such as respiration, photosynthesis, and nitrogen fixation. Although Fe is a ubiquitous element in circumneutral and well oxygenated environments it is found mostly in Fe(III) oxidize forms that have very low solubility and are not readily available for plants. As a consequence, plants have evolved two main strategies to acquire Fe: a reduction-based strategy (Strategy I) in dycotiledoneous and monocotyledoneous non poacea plants and a chelation strategy (Strategy II) in graminaceous ones. Strategy I plants are able to uptake only Fe(II) from the soil, so they are forced to reduce Fe(III) before carrying it inside the cell. The reduction- based mechanism of Fe acquisition relay on the combined function of three plasma membrane proteins: H+-ATPase, Fe-chelate reductases (FCR) and Iron Regulated Transporter (IRT). Fe deficiency induces the activation of these root plasmalemma enzymes/ion transporter as well as many other morphological and physiological responses at root level aiming at facilitate the mobilization of Fe (Schmidt, 2003; Marschner et al. 1986). Inside the cell the accumulation of Fe(II) possess also limitations as it catalyzes the generation of hydroxyl radicals that are potent oxidation agents resulting in oxidative damage of cellular components such as DNA and lipids. Thus, plants have to deal with a dual effect of Fe: on one hand the metal reduced form Fe(II) is essential for life, but on the other hand free Fe(II) is toxic. Therefore, to ensure Fe acquisition and avoiding toxicity, plants have to tightly control uptake, utilization, and storage of Fe in response to its environmental availability. A further limitation for Fe availability occurs in alkaline environments such as calcareous soils. In fact, Fe deficiency is a common nutritional disorder in crops growing on calcareous soils which account for 30% of world cultivated lands. In calcareous soils high pH decrease even more dramatically the Fe solubility. Moreover, it was reported that the presence of high levels of bicarbonate negatively affects either the FCR activity and/or IRT (Lucena et al. 2007). 2

Fe deficiency manifests itself primarily as an interveinal yellowing of the leaves known as chlorosis. Chlorosis is related to a diminution in chlorophyll due to the lack of Fe and leads to substantial agricultural losses and decreases in nutritional quality of many crops of great economic interest. Crop chlorosis is generally a result of both limited Fe bioavailability and cultivation of susceptible genotypes (Hansen et al. 2006). Furthermore, plants are the primary source of Fe for humans therefore a decrease in the amount of Fe in agricultural products has important implications for human health. Understanding the mechanisms of Fe uptake and regulation provide useful insights for producing plants tolerant to low Fe availability and able to increase their Fe content.

1.1. Iron in the soil Fe is the most abundant element in the Earth as a whole and the fourth most abundant element in the crust. (Taylor and Konhauser, 2011). Iron can exist in a range of oxidation states, from –2 to +6, but the most common are Fe(II) (ferrous iron) and Fe(III) (ferric iron). In soils three main sources of Fe can be recognized: - Fe contained in minerals; - Fe in soil solution; - Fe bound to organic matter. Fe availability in soils depends on a complex relation of the above components. In primary minerals Fe can be found mostly as ferromagnesian silicates such as olivine, augite, hornblende and biotite (Schwertmann and Taylor, 1989). Primary minerals release Fe(II) and Fe(III) through weathering processes of dissolution and oxidation. Fe(II) rapidly oxidizes to Fe(III) in the presence of water and atmospheric oxygen. Furthermore, free Fe forms a range of oxides, hydroxides and oxyhydroxides such as hematite, goethite and ferrihydrite. Most soils are oxic environments so Fe is mainly 3

found as Fe(III) oxides that confer to the soil typical colors varying from brown to yellow-red. Fe(III) oxides are stable, precipitates and have extremely low solubility. On the contrary, soluble Fe(II) oxides forms are found in anoxic and reducing environments. In an aqueous or moist environment, exposed to oxygen from Earth's atmosphere, Fe(II) has a half-life of several minutes as it rapidly oxidizes to Fe(III) (Stumm and Morgan 1996). Fe oxides solubility is basically the result of the equilibrium between dissolution and precipitation processes. The hydrolysis of Fe oxides is fully dependent on the pH solution. As show in figure 1 the concentration of all Fe species at a physiological pH (6-7) is far below the amount of Fe required by plant for optimal growth that ranges from 10-4 to 10-9 M (Guerinot and Yi, 1994).

Figure 1. Solubility diagram of the different Fe(III) species obtained by hydrolysis an iron oxide (goethite) as a function of pH. Black lines indicates Fe(III) species, while the grey line designates Fe(II). On the top grey bands indicates the range of concentration require for optimal growth of plants. Dashed area represents the optimal concentration for microbes. (from Robin et al., 2008)

From figure 1 it can be seen that: - Fe solubility is inversely related to the pH of the soil solution; 4

- total soluble Fe from different species reaches a minimum at about pH 8, a value close to that of calcareous soils; - in most Fe species for each unit increase in pH the solubility decreases from hundred to thousand fold - Fe concentrations for optimal plant growth would be met at a very acidic condition that is a pH value of 3.5

Many other factors influence Fe solubility as redox conditions, mineral particle size, humic substances, chelating agent’s content and the presence of bicarbonate. Under oxidizing conditions the activity of the readily available Fe(II) species are higher than the Fe(III) species at physiological pH and its concentration. However when the redox potential (pe) drops by 1 unit Fe(II) hydroxides concentration increases by two orders of magnitude. As mentioned before, the Fe species concentrations are anyway too low to supply available Fe to plants (Lindsay and Schwab, 1982). Conversely, under reducing conditions, where pe is very low, the concentration of soluble Fe(II) increases reaching high levels and becomes toxic. Typical reducing environments are flooded soils. The particle size of Fe minerals much affected its solubility: smaller the size of the particles, greater is the solubility of the Fe species. This is important because in well oxygenated environments nanometric particle sizes of Fe main minerals are very abundant (Cornell and Schwertmann, 2003). Moreover particle sizes greatly affect the kinetic of dissolution of Fe oxides (Schwertmann, 1991) also in the presence of organic ligands (Kraemer, 2004). The presence of organic matter considerably influences Fe solubility by complexation or chelation of Fe(III). The organic ligands are organic molecules that can bind to, and form a stable complex with metals wherein the metal is surrounded by the ligand or ligands. The organic ligands are called chelators or chelating agents while the metal complex is called chelate. Chelating agents usually originate from the activity of soil microrganisms, plants exudates and humic substance. Many organic 5

molecules containing unsaturated bonds, oxygen, nitrogen or sulphur, are likely to play the role of chelators of transition metals. Van Hess and Lundström (2000) have reported that more than 95% of Fe in soil solution was found to be organically bound. Compounds of very different nature act as chelating agents from low molecular weight organic compounds such as citric acid to complex substance like polyphenols or humic acids. Chelating agents play an important role in providing available Fe for plants through increasing its mobilization and bioavailability.

Calcareous soils contain sufficient CaCO3 and other carbonates. Because of the high values of soil solution pH,calcareous soils almost always fall in the alkaline range due to equilibrium reactions between Ca and bicarbonate. Although salinity and high percentage of exchangeable Na - (sodic soils) are alkaline, the soils dominate by HCO3 are the most alkaline ones (Läuchli and Grattan, 2012). The concentration of Fe expressed both on a dry weight leaf basis or on the amount of Fe per leaf frequently decreases in chlorotic leaves, although the Fe concentration can sometimes be the same or even higher in chlorotic leaves as compared with green ones (iron chlorosis paradox) (Morales et al., 1998; Römheld, 2000; Nikolic and Römheld, 2002). The presence of free soil carbonate promotes the formation of poorly soluble Fe phosphates impairing the availability of Fe. The solubilization of CaCO3 forms in calcareous soil solution leads to a high concentration of - HCO3 ions. It was reported for sunflower, soybean and cucumber that - addition of HCO3 at neutral pH could result in an inhibition of the activity of the key plasmalemma enzyme (FCR) involved in Fe uptake (Toulon et al., 1992). On the contrary it was demonstrate that Fe reducing capacity - increases when HCO3 was added at high pH values (Dofing et al., 1989; Romera et al., 1992). Despite contrasting findings that have been - obtained for different species it is clear that between HCO3 and Fe uptake mechanism an interaction occurs. Thus, in calcareous soil Fe unavailability is not only due to high pH and buffering conditions but also - to an interaction of HCO3 ion with the cell biochemistry. In Harmsen et al. (2005) bioavailable Fe has been defined “as the portion of total Fe that can be easily assimilated by living organisms.” As 6

mentioned above Fe bioavailability depends on many different factors that include inorganic and organic chemical processes, physical and biological processes. Furthermore it must be taken into account that different organisms vary in their acquisition pathways and capabilities, other than usually perform strategies to inhibit the growth of other species limiting nutrient competition (allelophaty). Variation between organisms may also result in differences of the soil areas in which each organism interacts with its environment, the so-called bioinfluenced zone, thereby altering the availability of the nutrient. For plants, this bioinfluenced zone typically corresponds to the rhizosphere (Harmsen et al., 2005). Soil microbial activity plays an important role in favouring plant Fe uptake. Typically soil microbes produce siderophores that is, relatively small molecules that have high affinity and chelate Fe. These effective chelators increase Fe bioavailability for plants. In fact, it has been widely evidenced that Fe bioavailability depends on the plant/microbe mutualistic interactions. Through root exudations plants provide a plenty of organic substances into rhizosphere supporting microbial communities that produce siderophores. A quite complex interaction settles among plant and microorganisms that lies in the equilibrium between competition for Fe, as plant and microorganism need it for growing, and enhancing Fe bioavailability (Lemanceau et al., 2009). Recent studies have suggested that in Fe deficiency treatments the number of microbes that produce siderophores increase as a result of the enhanced root exudation of phenolics by the plants (Jin et al., 2007).

1.2. Iron in plant physiology The biological importance of Fe lies on its electronic structure which can undergo reversible changes of its oxidation state in a wide range of redox potentials (pe). This ability confers to Fe a central role in all those biochemical reactions that involve transfer of electrons and variations of redox potential, as well as bind in a reversible way many ligands. The common biological ligands for Fe are oxygen, nitrogen and sulfur with which it form complexes of coordination. 7

The major classes of Fe-containing proteins are: - Fe-containing heme proteins; heme is a prosthetic group composed of a porphyrin ring structure with a central Fe2+ atom. There are many heme enzymes that catalyzed crucial biological processes such as DNA synthesis, chlorophyll synthesis, sulfur assimilation, ethylene synthesis. Heme is also found in cytochromes of the respiratory and photosynthetic chains, catalases, superoxide dismutase, oxidase and peroxidases that are involved in detoxification of Reactive Oxygen Species (ROS). - Fe-S proteins that are characterized by the presence of Fe-S clusters. In these clusters Fe is bond to S in different arrangement (Fe-S, 2Fe-2S, 4Fe- 4S, 3Fe-4S proteins) that are special redox centers. These proteins participate in various oxidation-reduction reactions. Fe-S proteins could accomplish also catalytic functions as aconitase which catalyzed the transformation of citrate in isocitrate. - proteins for Fe storage; Ferritin is a globular protein that can store from 2000 to 4500 atoms of Fe(III). Fe is almost never found in free form inside the cell. Once absorbed by epidermal or cortical root cells it is complexed with organic acids mainly citric and malic acids or other organic molecules which prevents Fe precipitation and avoid oxidative damages. In fact, once inside the cell, the redox capabilities of Fe can also be the basis of potential toxicity resulting from the Haber-Weiss-Fenton sequence that leads to the generation of hydroxyl radical (OH •) subsequent to the formation of - superoxide (O2 •) following the one-electron reduction of dioxygen (O2) by ferrous iron:

Figure 2. Haber-Weis-Fenton sequence. Fe(II) can react with O2 to form hydroxyl radicals and other reactive oxygen species. The hydroxyl radical is highly reactive and can damage virtually all types of macromolecules: carbohydrates, nucleic acids (mutations), lipids (lipid 8

peroxidation) and amino acids. The accumulation and transport of ionic Fe is improbable as Fe has a low solubility at physiological pH. The acquisition of Fe starts in the apoplast of the root epidermal cells. Fe inside the apoplast must undergo a reduction step before being transported through the plasmamembrane. It has been observed that as much as 75% of Fe in the roots is precipitated in the apoplast as hydroxide or phosphate salts (Bienfait et al. 1985) forming an apoplastic Fe pool. In addition Fe is attached to the apoplast since the negatively charged carboxyl groups of the cell walls serve as a cation sink. This Fe pool becomes important under Fe deficiency condition. It has been demonstrated that apoplastic Fe decreases when plant are grown under Fe starved conditions suggesting also the involvement of root exudates in facilitating Fe mobilization (Zhang et al. 1991, Jin et al. 2007). Once inside the cell Fe is bound by chelating agents to prevent its oxidation and as a consequence its precipitation and generation of hydroxyl radicals. Even if many organic acids and amino acids are potentially Fe chelators, NA seems to have a privileged function as Fe- chelator (Hell and Stephan, 2003). Fe–chelator complexes then move through the symplast into the stele along the diffusion gradient. At the pericycle, Fe is loaded into the xylem, and moves towards the shoot through the transpiration stream. When Fe is release into the xylem it is generally agreed that is reoxidized to Fe(III) and it is probably transported in the xylem as Fe(III)-citrate (Rellán-Álvarez et al. 2011). The mechanism by which Fe is unloaded from the xylem to the leaf tissues is not yet clear, but it has also to be mediated by transporters (Fe-citrate, Na-Fe or other complexes) and the photoreduction of the Fe-transporters seems to play an important role (Bienfait and Scheffers 1992). In leaves the plasmalemma activity of FCR has been demonstrated supporting the opinion of an enzymatic Fe reduction. The distribution of Fe in leaf cells is again most likely mediate by NA. To prevent the toxic effect of overload Fe in the leaf, Fe is stored in ferritin. Different groups of plant have very different optimum requirements of Fe from 0.5 to 50 ppm in higher plants. The general Fe content of green 9

plants tissues is of about 2 μmol g-1 of dry matter (Marschner 1995) which is quite lower in comparison of macronutrients, but represent the highest quantity among micronutrients.

1.3. Iron acquisition in dycotiledoneous plants: the Strategy I As pointed out before to face the problem of acquiring Fe dycotyledoneous and monocotyoledoneous non poaceae plants activate a reduce-based mechanism performed by, the so called Stategy I In general, Strategy I comprises physiological, developmental and metabolic reactions, adapting the plant to changing levels of available Fe sources. In Strategy I two main specific processes are involved, the reduction of Fe(III) chelates at the root surface and the transport of Fe(II) across the root plasma membrane, as these plants can only take up Fe(II). Other non specific mechanisms involved in Strategy I are the extrusion of H+ and exudation of organic acids and phenolics into the rhizosphere. Moreover, depending on Fe availability, morphological and architectural root changes accompanied the activation of the previous mechanism.

Figure 3. Schematic representation of Strategy I mechanisms for Fe acquisition. IRT= Iron Regulated Trasporter; FCR= ferric-chelate reductase; H+-ATPase= proton pump ATP dependent; PEZ= phenolic efflux zero transporter; CH= chelator 10

1.3.1. Reduction of Fe(III) chelates: a pre-condition to Fe acquisition. In all plants investigated so far FCR activity increased in response to Fe starvation and it has been observed that its activity can achieve an increased of as much as 10-20 fold respect to plants grown in complete nutritive substrates (López-Millán et al. 2000 and references therein). The transmembrane FCR acts transferring electrons through the plasma membrane that reduce Fe(III) to Fe(II) in the apoplast thus permitting its transport into the cell. A FCR responsible of the reduction step of Fe(III) to Fe(II) has been elucidated from the identification of the gene, FRO2, in Arabidopsis thaliana (Yi and Guerinot, 1994; Robinson et al. 1999). FRO2 belongs to a superfamily of flavocytochromes that transport electrons across membranes. As shown in figure 4 FCR is constituted by 8 transmembrane helices 4 of which present structural homology with the flavocytochrome b family. An important water soluble domain is localized between helices VIII and IX inside the membrane and contains NADPH, FAD and oxidoreductase activity. Between helices V and VII, probably two heme groups, are bind to conserved histidines. Flanking this assemble is located in the outer side of the membrane helix IV while in the inner side there is helix VI. The structural homology with flavocytochrome b suggest a similar mechanism of electron transport: NADPH is oxidized in the cytoplasm and electrons are transported through a flavine up to heme groups, when electrons arrive to the external surface of the membrane the reduction of F(III) to the Fe(II) occurs. 11

H histidine

heme group

Figure 4. Topology model of FRO2 take from Schagerlöf et al. (2006). Arrows indicate the localization of different fusion points.

In Arabidopsis other genes encoding isoforms of FCR were identified. Eight genes have shown conserved the domain where FAD and NADPH are bound to. Each isoforms seems to be expressed preferentially at different levels in the plant (leaves, roots, in photosynthetic tissues). These isoforms do not compensate from one another. FRO2 is responsible for Fe reduction at root level and mutants that lack in FRO2 gene do not show any FCR activity induced in other isoforms. Genes encoding for FCR has also been identified in other Strategy I species as pea, tomato and cucumber, their expression varies among species suggesting that FROs may have roles in Fe distribution in the plant (Jeong and Connolly, 2009 and references therein). In agreement with the idea that reduction of Fe is the rate-limiting step in Fe uptake by Strategy I plants (Grusak 1990), overexpression of FRO2 confers tolerance to growth on low Fe media. Moreover, overexpression of Arabidopsis FRO2 in soybean resulted in enhanced root ferric reductase activity and tolerance to Fe deﬁciency induced chlorosis (Vasconcelos et al. 2006). 12

1.3.2. Fe(II) transport inside the root cell. Once reduce Fe(II) is transported into the root by means of an IRT generally localized in the plasmalemma. The IRT was first discovered as an homolog of a wide family of multigene metal transporters (ZIP, zinc- regulated transporter iron-regulated transporter like protein) in Arabidopsis (IRT1), and even if it can transport other divalent metals like Mn, Zn, and Cd shows a high affinity for Fe (Kobayashi and Nishizawa, 2012). IRT1 plays a pivotal role in the regulation of plant Fe homeostasis, as demonstrated by the severe chlorosis and lethality of an irt1-1 knockout mutant (Vert et al. 2002, Henriques et al. 2002). Consistently, IRT1 gene is highly expressed in epidermal cells and the underlying cortex of Fe starved roots. Hence the Fe absorption dependent on IRT1 permits proper growth and development under iron limited conditions (Vert et al. 2002). The Zip family transporters in Arabidopsis contain 15 members (including IRT1) that can be divided in 4 groups according to the alignment of the predicted amino acid sequences. The high variety of transporters in Arabidopsis have been explained as a consequence of the high number of different membranes that cations have to cross to be distributed through the plant, suggesting that different ZIP protein could have different localizations and specific functions. Furthermore, it is not to be excluded that many ZIP proteins could exhibit functional redundancy (Mäser et al. 2001). IRT2, for example, is very similar to IRT1 in its aminoacidic sequence, it is also expressed in the external layer cells of Fe starved roots. However, it was observed that up-regulation of irt2 gene in knockout mutants of irt1 under Fe deficiency conditions was not sufficient to recover from the chlorotic phenotype (Vert et al. 2002). The predicted topology of most ZIP transporters has eight transmembrane domains and a similar orientation such that the amino and carboxy terminal ends are located on the extracellular surface. The difference of the various proteins is due to different lengths of the loop region (called variable region) between domains III and IV (Eide and Guerinot 1998). ZIP transporters have no similarities to other families of metal transporters. 13

Plants induce or repress various genes related to Fe homeostasis in response to Fe deficiency or Fe surplus. The molecular mechanism of control expression of IRT1 has been elucidated quite recently. IRT1 gene expression in root epidermis is transcriptionally promoted by a transcriptor factor know as FIT (Fe-deficiency-induced transcription factor) in Arabidopsis and FER in tomato which are positive regulators of root Fe deficiency responses (Colangelo and Guerinot 2004, Ling et al. 2002). FIT/FER play a decisive role in positively regulating various Fe deficiency inducible genes, including IRT1 and FRO2. Since FIT overexpression did not induce downstream genes under Fe sufficiency conditions it was suggested the existence of interacting partners that are expressed or activated as Fe deficiency response (Colangelo and Guerinot 2004, Jakoby et al. 2004). It was observed that Fe deficiency responses are transcriptionally regulated by a coexpression of different genes with FIT. IRT1 and FRO2 expression showed also a post-trascriptional regulation as the restoration of a Fe sufficiency medium induced a reduction in IRT1 protein accumulation and FRO2 activity in roots (Connolly et al. 2002). A work has reported that monoubiquitination of IRT1 at two lysine residues controls the subcellular localization, vacuolar sorting and degradation (Barberon et al. 2011). Many other studies have reported a large variability in genes involved in Fe deficiency depending on the specific response, function and localization. Other transcription factors identified are POPEYE (PYE) and BRUTUS (BTS) that play an important role at root morphology and growth level. Both PYE and BTS may act inversely; PYE regulates positively growth, elongation and swelling under Fe deficiency conditions while on the contrary BTS may repress them (Long et al. 2010). Other group of transporters belonging to the NRAMP (natural resistance-associated macrophage protein) family of integral membrane proteins was found in Arabidopsis. It was demonstrated the involvement of atNRAMP proteins in divalent metal transport (Curie et al. 2000) and their localization either in the plasma membrane and intracellular vesicles (Kobayashi and Nashizawa, 2012). Fe regulation and homeostasis involves a large number of genes that 14

expressed themselves at different localizations and with different modes; multiple pathway signaling and negative feed-backs loops. This complexity might confer the necessary flexibility to cope with an ever changing environment.

1.3.3. Acidification of the rhizosphere: the proton pump H+-ATPase In strategy I plants, Fe deficiency is generally associated with an increased extrusion of protons mediated by plasmalemma H+-ATPases. In Arabidopsis 12 isoforms of H+-ATPase are known from which AHA2 is involved in rhizosphere acidification whilst AHA7 seems related with the radical hairs development. The active extrusion of H+ is implicated in mineral nutrition among many other physiological functions such as control of the stomatal aperture, cell elongation, plant development, organ movement, and intracellular pH homeostasis, although evidence for the direct involvement of H+- ATPases in some of these roles is scarce (Sondergaart et al. 2004). In particular, regarding Fe nutrition the activation of this enzyme constitutes a key mechanism to Fe uptake by non-graminaceous plants and is tightly interrelated with the processes described in the previous paragraphs. The plasma membrane H+-ATPase is a universal electrogenic H+ pump, which uses ATP as energy source to pump H+ across the plasma membranes into the immediate vicinity of root surface (apoplast and rhizosphere). In general, the key function of this enzyme is to keep pH homeostasis of plant cells and generate an H+ electrochemical gradient providing the driving force for the active influx and efflux of ions and metabolites across the plasma membrane. Uptake of cations into plant cells is thus driven by ATP-dependent proton pumps. The resulting proton motive force typically comprises a membrane potential of about −150 mV, and a pH difference of 2 units (which contributes to another −120 mV to the proton motive force) (Mäser et al. 2001). As protons accumulate outside the cell, the pH of the apoplast decreases to reach values of 5–6 that are markedly more acidic than the cytoplasmic pH (Palmgren 2001). The acidification of the apoplast and the rhizosphere 15

is an essential step in Strategy I species since the solubility of Fe increases up to 1000-fold for each unit pH decrease. Thus, this process can have a huge impact on Fe activity in the close proximity of the roots (Olsen et al. 1981). The extrusion of H+ promotes the solubilization of Fe(III) and also balances the negative charges of the cell wall preventing repulsions of chelates. The H+ extrusion has also been associated to morphological changes in roots (López-Millán et al. 2000). Plasma membrane H+-ATPases are found throughout the plant in every cell type investigated so far. However, certain cell types have much higher concentrations of H+-ATPases than others. In general, cell types with abundant H+-ATPases are specialized in an active adsorption of solutes from their surroundings. In some strategy I species the increased acidification of the rhizosphere under Fe deficiency conditions occurs because an increase in protein abundance that are predominantly localized in epidermal cells being differentiated as transfer cells (Dell’Orto et al. 2002). Plant plasma membrane H+-ATPases is a single polypeptide of around 100 kDa that belongs to the large P-type H+-ATPases of cation pumps (Palmgren 2001). The enzyme has about 20% of its mass in the membrane, less than 10% is facing the non-cytoplasmic side and the largest mass is in four cytosolic domains, altogether accounting for about 70% of the mass (Palmgren and Harper 1999). The primary structure of Arabidopsis AHA2 predicts ten transmembrane helices (figure 5). In the cytoplasmic region there are 3 well distinct domains: A, the actuator domain; P, the phosphorylation domain and N, the nucleotidic binding domain that is fused with the P domain and contains the ATP binding site. Furthermore, there is a C-terminal domain (R) that exerts a post-translational auto-inhibitory regulation. In the inactive state the R-domain might indeed be close to or partially folded on the membrane and the activation of the enzyme depends on the phosphorylation of the penultimate Thr residue and the subsequent binding of 14-3-3 proteins that causes the displacement of the domain, removing the inhibition. The H+-ATPases are encoded by a gene family of about 10 members in 16

Arabidopsis thaliana and other species (Arango et al. 2003). Depending on the gene, expression is either restricted to particular cell types or widespread in the plant, with the possibility of more than one gene being expressed in a given cell type at the same developmental stage, thus excluding the characterization of a single isoform from plant material. A study carried out in cucumber has demonstrated a transcriptional regulation of a root plasma membrane H+-ATPase (Santi et al. 2005).

Figure 5. Schematic presentation of the AHA2 plasma membrane H+-ATPase. The various domains (A, P, N, and R) of the enzyme are indicated by colored residues (from Palmgren 2001)

Not all Strategy I species perform a high acidification and in those species in which acidification is very scarce the response depends on many factors such as the balance of cation/anion uptake, the composition of root exudates and the type of nitrogen nutrition. In conclusion, plants contain a complex regulation network of genes which encode uptake, chelation, transport, sub-cellular distribution and storage of Fe. Understanding these processes is the prerequisite for their manipulation in order to breed in the future high-quality nutritious crops.

1.3.4. The role of exudates in Fe acquisition Root exudates are considered as one of the main root products, which are well known to influence nutrient solubility and uptake when plants are subject to stress. Root exudates are involved in plant nutrition through direct or indirect mechanisms. In the first case, some compounds act directly on nutrients making them more available while, in the second case, exudates act on soil microbial communities generating mutualistic associations and determining the structure of microbial community in its area of influence. The quantity and quality of root exudates depends on the specie, the age of an individual plant and external factors like biotic and abiotic stress. The composition of root exudates can be complex, and often ranges from mucilage, root border cells, extracellular enzymes, simple and complex sugars, phenolics, amino acids, vitamins, organic acids, nitrogenous macromolecules such as purines and nucleosides to inorganic or gaseous − − + molecules such as HCO3 , OH , H , CO2 and H2. Many of these compounds have metal reductant or/and chelating abilities and can enhance Fe availability in the apoplast and rhizosphere (Ohwaki and Sugahara, 1997, Jin et al. 2007, Cesco et al. 2010, Rodríguez-Celma et al. 2011, Mimmo et al. 2012). These organic compounds can often be divided in two main classes: LOAs, which include amino acids, organic acids, sugars, phenolics and an array of secondary metabolites; and high-molecular weight compounds like mucilage and proteins. The exudation of a vast array of compounds is an important metabolic feature of plant root considering that a high portion of root carbon (70%) can be release into the rhizosphere (Neumann and Römheld 2007). Root exudation of various chemical molecules into the rhizosphere is largely dependent on the nutritional status of the plant, with some species exuding organic acid anions in response to P and Fe deﬁciency. The accumulation of low molecular weight organic acids (LOAs) has been recorded in many species as a response to Fe deficiency and may be involved in solubilization of iron from the soil. Landsberg (1981) has demonstrated that mono and dicots plant species released much more organic acids. The phenolic compounds are also important components of 18

root exudates and among their numerous functions they enhance the availability of iron to the roots (Julian et al. 1983, Jin et al. 2007, Cesco et al. 2010). In Fe deficiency conditions it has been demonstrate that dicots accumulate and exudates organic acids, mainly malate and citrate (de Vos et al. 1986, López-Bucio et al. 2000, Abadía et al. 2002). Organic acids due to their carboxylic groups can chelate metals in soil solution and thus are involved in mobilization and uptake of nutrients such us P and Fe. Furthermore when soil pH is high, as in calcareous soils, mobilization of Fe(III) by malate and citrate are very slow as the chelates they form are quite instable (Jones et al. 1996). However, in that condition it was suggested a coordinate action between organic acids and the acidification performed by the H+-ATPase (Jones, 1998). Several reports have indicated that cations were simultaneously released during the excretion of organic acids from roots. For example, wheat and lupin released K+ (Ryan et al. 1995) and H+ (Neumann and Römheld 1999) during the excretion of malate and citrate, respectively. Cation transport might be necessary for maintaining a transmembrane electrical potential difference during the release of organic acids (anion). LOAs are likely to cross the plasma membrane as multivalent anion. The pathways by which organic acids cross the plasma membrane of root cells are not well characterized and little is known of the molecular mechanisms that regulate the exudation of organic acids from roots. It has been suggested two main mechanisms of transport through the plasma membrane: diffusion as the gradient is favorable and anion channels that seems are directly related with the proton extrusion. Recently, a citrate-permeable channel in the plasma membrane of proteoid roots from white lupin has been characterized (Zhang et al. 2004). Many phenolic compounds have been identified in root exudates (Dakora and Phillips 2002, Jin et al. 2007, Cesco et al. 2010). Lan et al. has recently observed in a proteomic analysis a strong induction in phenylpropanoid pathways in Arabidopsis under Fe deficiency conditions. Phenolic compounds exhibited multiple functions in root exudates. Regarding Fe nutrition Jin et al. have demonstrate their role in mobilization of Fe apoplastic deposits (Jin et al. 2007). 19

1.3.5. Morphological changes induced by Fe deficiency The bioavailability of nutrients in the soil solution influences root growth, root proliferation and speciﬁc functional responses that in part depend on the prevalent nutrient status of the plant. Nitrogen (N), phosphorus (P), iron (Fe) and sulfur (S) are among the nutrients that have been reported to alter post-embryonic root developmental processes. Changes in root architecture can mediate the adaptation of plants to soils in which nutrient availability is limited by increasing the total absorptive surface of the root system. The development of root systems is usually highly asymmetric and reﬂects the ability of roots to adjust their growth and development to environmental factors (Forde and Lorenzo 2001). Furthermore, the morphological response is species specific even if a general trend could be drawn the variability among different species could be very high. Low Fe availability induces morphological changes both at macroscopic and microscopic level. Changes observed in root epidermal cell under Fe deficiency include an increase in root hair by modulating the length, position and abundance. Root hairs are the extensions of single epidermal cells and comprise as much as 77% of the total root surface area of cultivated crops, forming the major point of contact between the plant and the rhizosphere (Parker et al. 2000) and are involved in root secretion of compounds. It was seen that root hairs in plants under Fe deficiency occupied zones that did not have this structures in sufficient Fe conditions (Schmidt et al. 2000). Moreover, low Fe availability frequently leads to the formation of branched root hairs (Müller and Schmidt 2004) through a signaling cascade that probably involves auxin and ethylene (Schmidt et al. 2000, Schmidt and Schikora 2001). P deficiency induce similar changes in epidermal cells but their responses are mediated by different signal transduction pathways (Schmidt and Schikora 2001) In many species it was observed a subapical swelling as a response to low Fe availability that has been associated with an increase activity of the tips to acquire Fe. In Strategy I plants it was also found the development of rhizodermal transfer cells (Schikora et al. 2003; Santi and Schmidt 20

2008). Transfer cells are found in all taxonomic groups and have unique structural features: an invaginated secondary wall coated by the plasma membrane leading to an up to 20-fold area enlargement and is enriched in a set of transporters. Abundant mitochondria are also disposed near the plasma membrane of transfer cells. In these cells it was found a high metabolic activity that has been attributed to an increase activity of the Fe acquisition mechanism. Changes in root elongation and in the number of lateral roots were observed in Fe deficient roots. In a recent work on Arabidopsis it was point out that symplastic content of Fe triggers the local elongation of lateral roots by inducing an Auxin promoter. The changes in root morphology are directly related with hormone regulation. All root modifications previously pointed out are oriented to increase the contact surface of the root with the soil, its primary source of nutrients. The study of root system architecture modifications provides interesting inputs regarding the tight relationship between root development and soil resource. Root system architecture (RSA), the spatial configuration of a root system in the soil, is used to describe the shape and structure of root systems. Its importance in plant productivity lies in the fact that major soil resources are heterogeneously distributed in the soil, so that the spatial disposition of roots will substantially determine the ability of a plant to ensure edaphic resources. Therefore, studies revealing the extent and nature of the genetic variation of RSA have profound implications for improving water- and nutrient-use efficiency of crops or for enhancing their productivity under abiotic stresses or suboptimal soil conditions.

1.4. The Iron deficiency in crops According to FAO 39% of rural land areas are affected by mineral deficiency and low fertility that constrain crop production (Cramer et al., 2011). Iron deficiency is a problem in crop production worldwide but particularly in plants grown on calcareous soils (Vose, 1982). Calcareous soils represent the 30% of the earth land surface. Their total extent has 21

been estimated by FAO at 800 million hectares worldwide 57 million hectares of which in Europe. In the near future to cope with the increasing demand of food, agriculture must be extended to marginal areas, many of which include calcareous soils.

Figure 6. Soybeans varietal differences in iron deficiency chlorosis tolerance.

Iron deficiency in plants causes a nutritional disorder that results in dramatic yield losses and decreases the nutritional quality of crop products. The main Fe deficiency symptom in plants is chlorosis that consists in the yellowing of top leaves in the intervenial blade zones. Iron deficiency symptoms manifest themselves in the younger leaves due to the relative immobility of Fe in the plants. When Fe nutritional deficiency persist the leaves yellowing extends to the veins and the new leaves appears completely yellow. Low Fe availability results in substantial crop losses and reducing quality of crop products. Many differences in genotypic characteristic exist among crop species, and also among varieties of the same species, regarding their responses to low Fe availability. These differences are mainly base on the capacity to mobilize and acquire Fe from the rhizosphere. Crop Fe deficiencies have been reported for many plant species and geographical regions. The most frequent problems correspond to the cultivation of sensitive crop species in arid and semi-arid regions with calcareous soils for crop such as soybean (Glycine max), peanut (Arachis hupogaea), dry bean (Phaseolus vulgaris), sorghum (Sorghum bicolor) and rice (Oryza sativa). Fe deficiencies have also been reported in corn (Zea mays), wheat (Triticum aestivum) and oat (Avenae sativa) but only in particular conditions. The following crops have been also found to be 22

especially responsive to Fe low bioavailability: alfalfa, asparagus, barley, beans (white), beets, broccoli, brussel sprouts, cabbage, cauliflower, celery, peach, pear, kiwifruit, the grapevine riparia, citrus, grass, oats, peanuts, rye, spinach, strawberry, sudangrass, tomato, and turf. In areas where Fe deficiency is manifested, the deficiencies generally occur in patches rather than uniformly across a field. Chlorotic patches often occur in fields where high soil pH is homogeneous, indicating that alkalinity is not the only factor controlling the availability and uptake of Fe (Hansen et al. 2006). Many interactions exist between different mineral especially between P and Fe. In fact the appearance of chlorosis depends on the P/Fe ratio rather than the single amounts of one or another nutrient. Because plants are a primary food source for humans, the nutritional state of plants is also of central importance to human health. In particular, Fe content in plants becomes important in those populations whose diet is primarily vegetal. Understanding of Fe homeostasis has been successfully applied to generate crops that are tolerant of Fe deficiency or whose edible parts are more nutritious. Creating designer crops with enhanced nutrient uptake could also aid agriculture to reduce the need for fertilizer application decreasing consistently the managing costs of agriculture.

1.5. Wild plant strategies to low nutritional deficiencies: the case of iron deficiency A Plant strategy is defined as a set of interlinked adaptations that arose as a consequence of natural selection and promotes growth and successful reproduction in a given environment (Craine, 2009). The traits associated with plant resource strategies are important for the vegetative growth of individual plants and determine the acquisition, allocation, and loss of resources that support successful growth under a particular set of environmental conditions. These traits range from biochemistry of the cell to tissue construction, to plant effects on the abiotic environment. Many plants are well adapted to leave successfully in low-nutrient 23

environments. The traits observed in plants performing a low-nutrient strategy are related with: - increasing the acquisition of nutrients; - decreasing the requirement of nutrients; - decreasing losses of nutrients. Increasing the acquisition of nutrients implies the activation of strategies of mining and scavenging of nutrients. Mining strategies comprise all those mechanism oriented to mobilize the not yet available resources. In previous paragraphs the mining mechanisms that are activated by plants to make available the Fe present in the soil are presented: the reduction- based mechanism of Fe acquisition (par. 1.3.1-1.3.3.), the exudation of organic compounds (par. 1.3.4.), and the scavenging strategies that mainly consist in exploring the soil and hence directly related to root morphology (par. 1.3.5.). Decreasing nutrient requirements does not necessary mean decrease the nutrient requirement per plant but rather the reduction of nutrient to produce a given unit of biomass. Species that display low nutrient strategies shifts the nutrient flow to those activities considered primary while on the contrary all purposes of secondary importance to growth are restricted in the limited nutrient. In many wild species flowering and produce seed or produce resistant structures may have priority over vegetative growth. Decreasing losses of nutrients refers mainly to the turnover of biomass. Generally low nutrient strategy species reduce the loss rate of biomass having increase longevity of leaves and roots. These observations match well with physiological traits observed in non- crop plants that are plants that have been developed through natural selection of all coordinated adaptations of roots, leaves, and support structures. The acclimation of plants to abiotic stress conditions is a complex and coordinated response involving hundreds of genes. Study how plants sense and acclimate to abiotic stress conditions is crucial to develop plants and 24

crops with enhanced tolerance to abiotic stresses.

1.6. Parietaria judaica: a wild species tolerant to high calcareous environments. Parietaria judaica (L. 1753) – of which Parietaria diffusa (M. et K. 1823) or Parietaria ramiflora (Moench 1794) are synonyms – belongs to the Urticacea family. It is a wild perennial sinantropic plant.

A B C A

Figure 7. Parietaria judaica. A spontaneous growth, B and C detail of P. judaica morphology

Parietaria judaica stands 0.2 until 1.0 m tall, displaying variable habits depending on the environmental characteristics, from spreading to decumbent-erect. The leaves are alternate from lanceolate to oval and 2- 8 cm long. Both leaves’ surfaces are covered with fine white hairs. It reproduces sexually and asexually. Flowering season is up to ten month. Seeds germinate over a wide range of temperatures from 10 to 27°C, with 75% of germination seeds at 20°C in light and dark conditions. Seeds are dispersed by wind (anemochorous). Most scientific literature concerns studies on immunology and biomedicine due to its high pollen allergeny (Colombo et al. 2003; Barranca et al. 2010) and medical properties (Uncini Manganelli et al. 2005). 25

Studies in ethnobotanics have identified many active principles in Parietaria such as flavonoids. At the moment a detailed chemical characterization of Parietaria j. has not been carried out, anyway it is well known that it has a high content in KNO3 as well as polyphenols (Donnini et al., 2012). Even though it is consider an “indiffent plant” due to its ability to grow and complete successfully its life cycle in both acid and alkaline substrates, it constitute the widespread flora in calcareous hostile environments, as wall cracks exposed to sun without showing any sign of chlorosis. As a dicot, Parietaria encompasses all the inducible metabolic changes of Strategy I Fe efficiency (dell’Orto et al. 2003). However the extreme efficiency by which it adapts to stress conditions suggested a great metabolic flexibility. In particular the high production of phenols almost certainly plays an important role within its adaptive strategy.

1.7. Aim of the research The study of wild plants responses to calcareous conditions could provide interesting insights as they are well adapted to the environment. 30% of all cultivated soils are calcareous and considerable crop losses result from the cultivation in these types of soils. The natural adaptation of wild plants to the environment occurs on the entire plant at different levels so the adaptive response always involves different traits. More diversified and flexible is the response more possibilities of adaptive success are. This study focusses on the integrated responses of a wild calcicole plant Parietaria judaica to different alkaline conditions. The main purpose of the study is to characterized the diversified responses at morphological, physiological and biochemical level. Another goal is to distinguish the response due to the presence of a high concentration of bicarbonate in the growing medium from a response due only to a high pH of the growing condition. In this regard two different high alkaline buffer treatments were performed: one with CaCO3/NaHCO3 which mimics the calcareous environment and other with a high alkaline organic buffer (Tricine). The main Strategy I responses were recorded: reduction by means of the iron chelate reductase activity; acidification through the activation of the plasmalemma H+-ATPase; the content of low organic acids (LOAs) and phenolics compounds in root tissues and exudates. Activation of Strategy I requires an increase production of energetic metabolites as ATP and NAD(P)H+, LOAs, and secondary metabolites such as phenolic compounds. This requirement implies a general metabolic arrangement of the cell. Further responses to Fe starvation are concerned with alterations in root morphology and root architecture, often leading to an increase in the absorptive surface area. Depending on the species, such an increase can be achieved by the formation of extra root hairs, development of clusters of secondary lateral roots (proteoid roots), or the formation of transfer 27

cells in the rhizodermis. Morphological changes of the root system as a response to direct or induced Fe deficiency were archived using an image system analysis with the aim to identifying the patterns of morphological change. The study of the metabolic rearrangement in a wild species (Parietaria judaica) grown in different conditions of iron availability could provide interesting insights in order to recognize the adaptive traits of a spontaneous plant coping with a highly calcareous environment.

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Adaptive strategies of Parietaria diffusa (M.& K.) to calcareous habitat with limited iron availability* Silvia Donnini, Patrizia De Nisi, Damiano Gabotti, Liliana Tato, Graziano Zocchi *published in Plant, Cell and Environment, 2012 35(6):1171–1184 36

ABSTRACT The study of native plants growing in hostile environments is useful to understand how these species respond to stress conditions. Parietaria diffusa (M.& K.) is able to survive in highly calcareous soils and extreme environments, such as house walls, without displaying any chlorotic symptoms. Here, we have investigated the existence of Strategy I complementary/alternative mechanism(s) involved in Fe solubilization and uptake and responsible for Parietaria’s extraordinary efficiency. After assessing the specific traits involved in a calcicole-behaviour in the field, we have grown plants in conditions of Fe deficiency, either direct (-Fe) or induced by the presence of bicarbonate (+FeBic). Then, the growth performance, physiological and biochemical responses of the plants were investigated. The study shows that in Parietaria +FeBic, the classical responses of Strategy I plants are activated to a lower extent than in -Fe. In addition, there is a greater production of phenolics and organic acids that are both exuded and accumulated in the roots, which in turn show structures similar to ‘proteoid-like roots’. We suggest that in the presence of this constraint, Parietaria undergoes some metabolic rearrangements that involve PEP-consuming reactions and an enhancement of the shikimate pathway. Key-words: bicarbonate; calcareous soils; Fe deficiency; organic acids; phenolics.

2.1. Introduction In an ecosystem, the colonization by a specific plant species affects biodiversity. However, there are few studies about the physiological properties of species that determine their success during ecological competition and therefore their environmental distribution. The chemico- physical characteristics of the soil are often important factors that strongly control plant distribution and growth. It is well known that the presence of bicarbonate and high pH in the soil cause a diminished bioavailability of some nutrients, mainly Fe, inducing in plants a common nutritional disorder named chlorosis (Lindsay 1984). Given that more than 30% of the soils in the world are calcareous, it is

quite common that crops must face this problem (Chen and Barak 1982). Plants show different susceptibility to soil factors inducing Fe deficiency, so that a different degree of stress and a great variability exists in response to Fe deprivation. A variability in Fe acquisition capacity has been found in spontaneous vegetation (Nelson 1992), and native plants can be assigned to Fe-efficient and Fe-inefficient classes on the basis of the presence and diffusion in pedological environments inducing a low Fe availability. In particular, the so called calcicole species seem to strongly colonize calcareous habitats better than other species (i.e. calcifuge plants), demonstrating a better efficiency in obtaining Fe under these unfavourable environments. In fact, it has been demonstrated that calcicole native dicotyledonous plants are more Fe-efficient than calcifuge ones, showing a higher Fe3+-chelate reductase activity, a response assigned to the Strategy I (Schmidt and Bartles 1998). Strategy I is a complex Fe uptake mechanism developed by all plants, with the only exclusion of the Poaceae, which belong to Strategy II (Römheld and Marschner 1986; Morrissey and Guerinot 2009; Abadía et al. 2011). This Fe uptake mechanism is based on the reduction of external Fe3+ to Fe2+ through the induction of a Fe3+-chelate reductase enzyme localized at the plasma membrane of the rhizodermal cells (Robinson et al. 1999; Waters, Blevins and Eide 2002; Li, Cheng and Ling 2004). Once reduced, the Fe2+ ion is transported inside the roots by a carrier (IRTs) belonging to the ZIP family of transporters (Guerinot 2000; Varotto et al. 2002; Vert et al. 2002; Henriques et al. 2002). The capacity to grow in Fe-poor environments is not restricted only to the induction of the two above mentioned mechanisms, but other activities can be induced during Fe deficiency stress. Amongst these, several biochemical and morphological adaptations occur in Strategy I plants. One of the most important is the enhanced capacity to decrease the rhizospheric pH through the activation of a plasma membrane-localized P-type H+-ATPase (Zocchi and Cocucci 1990; Palmgren 2001; Santi and Schmidt 2009). Proton extrusion is useful both to increase Fe availability by increasing the solubility of Fe compounds (Dell’Orto et al. 2000), as well as to set up a favourable transmembrane electrical potential (negative inside) for cation uptake. However, the induction of the reduction processes and the enhanced extrusion of protons under Fe deficiency needs an increased rate of

NAD(P)H and ATP regeneration, which is achieved through the acceleration of metabolism, in particular glycolysis. Amongst the metabolic activities which are increased under Fe deficiency, phosphoenolpyruvate carboxylase (PEPC) seems to play a pivotal role (Zocchi 2006 and references therein). In fact, the enzyme is active in several anaplerotic reactions to replenish tricarboxylic acid cycle intermediates, to provide carbon skeletons to sustain the synthesis of amino acids and for the pH-stat mechanism (De Nisi and Zocchi 2000; López-Millán et al. 2000; Zocchi 2006). In some Strategy I species also the release of organic compounds such as phenolics, flavins, sugars and organic acids could help in the solubilisation of Fe containing compounds (Welkie 2000; Curie and Briat 2003; Jin et al. 2007; López-Millán et al. 2009). Considering the reducing and complexing proprieties of phenolic compounds, it is widely accepted that the mechanism by which they can regulate Fe mobility in the rhizosphere might play an important role (Jin et al. 2007; Tomasi et al. 2008; Cesco et al. 2010). Recently, a few studies have explored the role of secondary metabolic pathways in plant response to Fe deficiency. An example is the shikimate pathway, which is responsible for the synthesis of phenolic compounds. Some of the key enzymes catalyzing this pathway include shikimate dehydrogenase (SDH), shikimate kinase (SK), and phenylalanine ammonia lyase (PAL) (M’Sehli et al. 2009; Lan et al. 2011). From a morphological point of view, the appearance of swollen tips, secondary lateral roots and root hairs increase the surface of contact between roots and soil, favouring the search and acquisition of nutrients (Landsberg 1996; Schmidt and Bartels 1996). Induction of these different responses, in addition to reduction and transport processes, would increase plant efficiency in solving the problem of Fe deficiency. The more a plant is able to diversify its response, the more efficient will be the result. In alkaline and calcareous environments, Fe solubility and availability are drastically reduced along with other nutrients (for instance P) (Guerinot Yi 1994; von Wandruszka 2006). This means that in these conditions, relatively low amount of soluble nutrients might be available for plant uptake and therefore for use in various physiological functions, affecting photosynthesis, growth, competition and survival of the species. However,

heavy calcareous sites are colonized by plants with morpho-physiological characteristics of resistance to these particular conditions. Parietaria diffusa, a spontaneous species also named “pellitory of the wall”, is a dicot belonging to the Urticaceae family, widespread in the Mediterranean area, United States and Australia. It is able to grow on walls, debris and substrates with a very high carbonate concentration without showing any Fe deficiency symptom (Fig. 1). For this reason Parietaria could represent a model plant to understand the ecophysiological traits determining the calcicole behaviour of plants and the mechanisms of resistance developed by them to face with these adverse conditions, particularly concerning Fe deficiency.

Figure 1. Spontaneously growing Parietaria diffusa (M&K).

In a previous work (Dell’Orto et al. 2003) we have characterized in part the biochemical and physiological responses of Parietaria under Fe deficiency conditions, typical of a calcareous habitat, in order to clarify whether it behaves as a Strategy I plant. In the present study some secondary responses to Fe deficiency were further investigated, with the aim to determine the existence of alternative or complementary mechanism(s) to the typical Strategy I, which may be responsible for Parietaria’s extraordinary efficiency in Fe acquisition. In particular, to identify the specific traits of tolerance involved in a calcicole-behaviour, an analysis on Parietaria plants collected directly from walls with

different pH values has been carried out. Then, Parietaria plants have been grown in hydroponics under Fe deficiency, either direct or induced by the presence of bicarbonate, and the morphological, physiological and the biochemical traits of the plants have been investigated.

2.2. Materials and methods 2.2.1. Plant Material

2.2.1.1. FIELD SAMPLING Parietaria diffusa (M.&K.) plants were sampled in three different sites near the University of Milan (Italy). Sampling areas were chosen a priori based on the presence of Parietaria as the unique species. Samples were collected by breaking the walls and the substrates surrounding roots were also collected and solubilized in distilled water in order to determine the pH (pH-meter PHM 240 pH/Ion Meter).

2.2.1.2. HYDROPONIC CULTURE Cuttings of Parietaria judaica were obtained from a mother plant and put in an aerated half strength nutrient solution for 1 week to radicate. Once rooted, plants were transferred to 10 L plastic pots (40 plants/pot) containing (i) full nutrient solution plus 100 µM Fe(III)-EDTA (+Fe), pH 6.2; (ii) full nutrient solution without Fe (-Fe), pH 6.2; (iii) full nutrient -1 solution with 100 µM Fe(III)-EDTA, 15 mM NaHCO3 and 0.5 g L CaCO3 -1 (+FeBic), which brought the pH to 8.3. Addition of 0.5 g L CaCO3 is useful to keep the bicarbonate concentration constant to approximately 15 mM throughout the experiment. The composition of the full strength solution was as reported in Donnini et al. (2010). Plants were grown for 9 days under different treatments in a growth chamber under 16/8 h light/dark regime, 27/21°C, 65-75% relative humidity and with a PPDF of 200 µmol m-2 s-1.

2.2.2. Collection of root exudates Root exudates were collected according to Gries et al. (1995). Five plants from each treatment were transferred to 250 ml of distilled water, with the pH adjusted to 6.2, containing 10 mg L−1 of Micropur™ to prevent decomposition of organic matter by microorganisms. Root exudates were collected over a 24 h period in a growth chamber under continuous aeration and freeze dried. The lyophilized material was resuspended in distilled water and filtered through a 0,45 m Millipore Millex-HN; both phenolics and organic acid concentrations were then determined. For each treatment five replicates were performed.

2.2.3. Determination of phenolic compounds Determination of phenolics concentration in the roots was performed using two different extraction solutions; distilled water or methanol. Sampled roots were homogenized in 1 volume (w/v) of extraction solution and the homogenate centrifuged at 10 000 g for 10 min. Extracts were filtered through a 0,45 m Millipore Millex-HN. The concentration of phenolics in both roots and exudates was determined spectrophotometrically at 750 nm with the Folin-Ciocalteau reagent according to the method of Swain and Hillis (1959), using gallic acid as a standard. For each treatment five replicates were performed.

2.2.4. Organic acids assay For the quantitative determination of organic acids, roots were collected, carefully rinsed in distilled water and homogenized in the presence of 5 mL of 10% (v/v) perchloric acid and centrifuged at 10 000 g for 15 min.

The pH was brought to 7.5 with 0.5 M K2CO3 to neutralize the acidity and to precipitate the perchlorate. Extracts were clarified by centrifugation at 15 000 g for 15 min. Citric and malic acid concentration in both roots and exudates was determined enzymatically, using specific kits from Boehringer Mannheim and according to the manufacturer’s instructions. The recovery of both organic acids was more than 90% as determined by

the use of an internal standard (Rabotti, De Nisi and Zocchi 1995). For each treatment five replicates were performed.

2.2.5. Biomass measurement and leaf chlorophyll determination Growth of shoots and roots was calculated as the difference between the end (9 d) and the beginning of treatments. FW and DW were determined at the end of treatments. Three independent experiments in triplicate (n=9) were performed. Chlorophyll concentration was determined according to Lichtenthaler (1987). For each treatment five replicates were performed.

2.2.6. Determination of Fe and P For the determination of apoplastic Fe, plants from each treatment (n=5) were transferred to a beaker with 0.5 mM CaSO4 under vigorous aeration. After 10 to 15 min, plants were placed with their root system in 40-ml tubes with 21 ml of 10 mM MES, 0.5 mM Ca(NO3)2, 1.5 mM 2,2’-bipyridyl

(pH 5.5) at 25°C. Tubes were covered with a cotton plug and N2 was bubbled through the solution. After 5 min, 1 ml of 250 mM Na2S2O4 was added from a syringe. The A520 of the solution (A520 of 1 mM Fe[bipyridyl]3 = 8.650) was determined on 2 ml aliquots as reported by Bienfait, van den Briel and Mesland-Mul (1985). After removal of apoplastic Fe the amount of Fe and P accumulated in the same plant was determined. For this, roots and leaves were collected separately, carefully rinsed with distilled water and oven-dried at 55°C, ground to powder and mineralized in 1M

HNO3. Final volumes were adjusted to 1 mL by 0.1 M HNO3. Fe was determined by ICP-OES (Sequential ICP-OES AX Liberty, Varian) and P by ICP-MS (Varian 820-MS).

2.2.7. Fe reduction and acidification in vivo Visualization and localization of Fe reduction and acidification activities of the roots were performed using the agar technique as reported by

Donnini et al. (2009). Root segments were extensively washed until the pH in the solution was stable at pH 6.1 and embedded in 0.75% (w/v) agar containing 100 µM Fe(III)-EDTA and 100 µM bathophenanthroline disulfonate (BPDS) for reduction activity or 0.006% bromocresol purple (pH indicator) for acidification activity. The experiment was repeated three times with the same results.

2.2.8. Fe reduction by phenolics exuded and accumulated in the roots The phenolics’ ability to reduce Fe(III)-EDTA was measured spectrophotometrically using the BPDS reagent (Chaney, Brown and Tiffin 1972). 75-100 g of phenolics prepared according to the method described above were incubated for 120 min in 1 ml of a solution containing 100 µM Fe(III)-EDTA and 100 µM BPDS in the dark at 26°C under shaking. The absorbance at 535 nm was determined as reported by Donnini et al. (2009). The experiment was repeated three times.

2.2.9. Preparation of plasma membrane vesicles Roots of 9-d-old plants were excised, rinsed in distilled water and homogenized in a mortar at 2–4°C in a buffer containing 50 mM MOPS-BTP pH 7.5, 330 mM sucrose, 5 mM EDTA, 1 mg mL-1 bovine serum albumin (BSA), 5 mM dithiothritol (DTT), 2 g mL-1 PMSF, 10 % (w/v) diethyldithiocarbamic acid sodium salt (DIECA). After filtration through four layers of gauze, the homogenate was centrifuged at 13 000 g for 15 min and the supernatant centrifuged at 100 000 g for 30 min to obtain a microsomal fraction. The plasma membrane enriched fraction was prepared using the two-phase partitioning procedure as reported by Larsson, Sommarin and Widell (1994). The enrichment in plasma membrane was determined assaying vanadate-sensitive H+-ATPase (EC 3.6.1.35) (PM), nitrate-sensitive H+-ATPase (EC 3.6.1.3) (tonoplast) and azide-sensitive H+-ATPase (EC 3.6.1.34) (mitochondria), respectively, according to Rabotti and Zocchi (1994).

2.2.10. H+-ATPase assay H+-ATPase activity in plasma membrane preparations was determined in a medium with the following composition: 250 mM sucrose, 50 mM KCl, 25 mM MOPS-BTP pH 6.5, 1 mM ATP, 0.25 mM NADH, 1 mM PEP, 15 μg mL−1 lactate dehydrogenase (EC 1.1.1.27), 30 μg mL−1 pyruvate kinase (EC 2.7.1.40), 0.015% (w/v) Lubrol and 10–20 μg plasma membrane protein (omitted in blanks). The reaction was started by the addition of 1 mM

MgSO4. NADH oxidation was followed at 340 nm. Three independent experiments in triplicate (n=9) were performed.

2.2.11. FC-R assay The NADH-dependent Fe(III)-reductase activity of plasma membrane preparations isolated from roots was determined at 26°C in a medium containing 250 mM sucrose, 15 mM Mops-BTP pH 7, 0.25 mM K3Fe(CN)6, 0.25 mM NADH and 0.01% (w/v) Lubrol and the reaction was started by the addition of 10-20 µg of plasma membrane protein (omitted in blanks). The absorbance change at 340 nm was monitored. Three independent experiments in triplicate (n=9) were performed.

2.2.12. Soluble protein extraction and PEPC assay Roots of 9-d-old plants grown under the different treatments were harvested, rinsed and homogenized in a buffer (1 mL g-1 FW) containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 10% (v/v) glycerol, 1 mM EDTA, 14 mM ß-mercaptoethanol, 1 mM PMSF and 10 μg mL−1 leupeptin. The homogenate was filtered through four layers of gauze and centrifuged at 13 000 g for 15 min and the supernatant was again centrifuged at 100 000 g for 30 min. Phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) was determined as in De Nisi and Zocchi (2000). The reaction was started by the addition of 20-50 L of soluble fraction (omitted in blanks) and the absorbance change at 340 nm was monitored. Three independent experiments in triplicate (n=9) were performed.

2.2.13. Western blot analysis of PEPC and H+-ATPase Soluble and plasma membrane proteins (15 µg) were loaded on a discontinuous SDS-polyacrylamide gel (3.75% (w/v) acrylamide stacking gel and 8% (w/v) acrylamide separating gel). After SDS-PAGE, western blot analyses were performed as reported in De Nisi and Zocchi (2000) for PEPC and in Dell’Orto et al. (2000) for the H+-ATPase, respectively. Two different antisera were used; one raised against the central domain of PM H+-ATPase of Arabidopsis thaliana (a kind gift from Dr R. Serrano) and a second one raised against a PEPC isoform of sorghum (a kind gift from Dr J. Vidal). The experiment was repeated three times with the same result.

2.2.14. Shikimate pathway enzyme extraction and assay For 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS, EC 4.1.2.15) 0.2 g fresh roots were homogenized in 100 mM potassium-phosphate buffer (pH 8.0) containing 1.4 mM β-mercaptoethanol. The homogenate was centrifuged at 15 000 g for 15 min at 4 °C and DAHPS activity was assayed on soluble fraction as reported by Sánchez-Rodríguez et al. (2011). Shikimate dehydrogenase (SDH) and shikimate kinase (SK) extraction were performed according to Díaz, Barceló and Merino (1997). SDH (EC 1.1.1.25) was assayed at 25°C by monitoring the reduction of NADP+ at 340 nm according to Chaudhuri and Coggins (1985). The assay mixture contained 20-50 L of soluble fraction (omitted in blanks) in 100 + mM Na2CO3 pH 10.6, 4 mM shikimic acid and 2 mM NADP . SK (EC 2.7.1.71) was assayed at 25°C by coupling the release of ADP to the oxidation of NADH using pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) as coupling enzymes according to Krell et al. (2001). Shikimate-dependent oxidation of NADH was monitored at 340 nm. The assay mixture contained 20-50 L of soluble fraction (omitted in blanks) in 50 mM triethanolamine hydrochloride/KOH buffer at pH 7.0, 50 mM KCl, 5 mM MgCl2, 1.6 mM shikimic acid, 5 mM ATP, 1 mM PEP, 0.1 mM NADH, 30 g mL-1 pyruvate kinase, and 15 g mL-1 lactate dehydrogenase.

The enzyme 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) syntase (EC 2.5.1.19) was extracted and assayed as reported by Forlani, Nielsen and Racchi (1992). EPSP synthase activity was measured in a medium containing 100 mM Hepes NaOH pH 7.4, 1 mM shikimate-3-phosphate (S3P), 1 mM PEP, 0.5 mM ammonium heptamolybdate and 60 L of enzyme. After incubation, the reaction was stopped by addition of a solution containing malachite green and samples were read at 660 nm against blanks in which S3P had been omitted. Phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was obtained by homogenizing 0.20 g fresh tissue in 15 mL of an extraction medium containing 20 mM ß-mercaptoethanol, 0.1 M sodium borate buffer pH 8.8 and 5% (w/v) PVPP. After filtration through four layers of gauze, the homogenate was centrifuged at 12 000 g for 20 min, 4 °C. The enzyme activity was determined by adding 1 mL of the protein extract (omitted in blanks) to a reaction medium according to Cahill and Mc Comb (1992). All the enzymes were assayed by three independent experiments in triplicate (n=9).

2.2.15. Protein determination Protein content was determined by the Bradford (1976) procedure using BSA as a standard.

2.2.16. Oxygen consumption Apical root segments (2 cm) were excised under water at room temperature from plants illuminated for several hours. Root O2 consumption rates were measured from the decrease in O2 concentration in an aqueous phase with a Clark-type O2 electrode (YS1 Analytical control) at 25° C. Calibration was made from the difference in signal between aerated water and Na-dithionite saturated water. The effects of KCN (2mM) and salycilichydroxamic acid (SHAM) (2mM) were determined as reported by Vigani, Maffi and Zocchi (2009). Five replicates for each treatment were performed.

2.2.17. Statistics All statistical analyses were conducted with Sigma-Stat® 3.1. Means were compared by t test at P< 0.05 in all cases. 2.3. Results 2.3.1. Organic compounds in roots and exudates

Table 1. Water and methanol soluble phenolics, malic acid and citric acid content in Parietaria roots and exudates. Plants were collected either from the field or from the hydroponic culture. Growth conditions are reported as in Materials and Methods section. Data are means S.D. (n = 5). In the case of significant differences (P 0.05) values are marked with different letters. water-soluble methanol- malic acid citric acid Sample phenolics soluble ( g g-1 FW) ( g g-1 FW) (mg g-1 FW) phenolics -1 (mg g FW) Parietaria in the field (mg g-1 FW) pH 7.19 3.54 0.29 c 7.85 0.17 b 596.91 8.15 c 188.06 5.37 b pH 7.78 3.79 0.14 b 8.17 0.07 b 635.83 5.27 b 224.30 9.22 b pH 8.3 4.40 0.21 a 8.22 0.18 a 696.96 7.23 a 387.21 10.15a Parietaria in hydroponic +Fe (pH 6.2) 0.13 0.02 c 0.17 0.02 c 343.71 11.46 c 75.05 9.68 c -Fe (pH 6.2) 0.39 0.03 b 0.67 0.05 b 370.22 12.18 c 366.67 4.31 b

+FeBic (pH 8.3) 0.93 0.07 a 3.77 0.03 a 1127.4 29.74 a 623.98 7.66 a Exudates in hydroponic +Fe (pH 6.2) 0.028 0.002 c - 1.68 0.21 c 0.33 0.09 c -Fe (pH 6.2) 0.033 0.003 b - 7.67 0.48 a 5.89 0.21 b +FeBic (pH 8.3) 0.045 0.005 a - 5.17 0.91 b 10.87 0.29 a

Table 1 shows the results for Parietaria roots sampled both in the field and in hydroponic culture. Concerning the first ones, we observed a correlation between the increase in pH of the substrates surrounding roots and phenolics accumulation in the root. A similar trend was also observed for malic and citric acid concentrations, which increased in the

roots when the pH of the growing medium increased. In Parietaria plants grown in hydroponic culture, the absence of Fe induced an accumulation of phenolics in the roots (about 3- and 4-fold when extracted in water and methanol, respectively). Also in the presence of bicarbonate, an increase in phenolics concentrations occurred in the roots (7- and 22-fold when extracted in water and methanol, respectively). Both malic and citric acid concentrations were found to increase (8% and 5-fold, respectively) as Fe in the hydroponic culture was removed. However, when bicarbonate was added to the +Fe solution, the concentrations of organic acids were significantly higher (3.3- and 8.3-fold increases for malic and citric acid, respectively). Concerning phenolics concentrations in the exudates, an increase in –Fe plants (+18%) and in plants grown in the presence of bicarbonate (+61%) was detected. The malic acid concentration in exudates of plant exposed to –Fe condition was significantly higher than in plants grown in the presence of bicarbonate, whereas for citric acid the major increase was found in the +FeBic treatment (Table 1). With respect to the relative controls, the malic acid concentration increased by 4.6-fold and 3-fold in –Fe and +FeBic treatments respectively, whereas citric acid concentration increased by about 18-fold and 33-fold in –Fe and +FeBic, respectively.

2.3.2. Biomass measurement Plant growth was influenced by the stress conditions and a significant decrease in shoot length was found both in the absence of Fe (-31%) and in the presence of bicarbonate (-55%). Root length also decreased when bicarbonate was added to the medium (-74%), while the –Fe treatment did not cause significant decreases in this parameter (Fig. 2A). The fresh weight (FW) of the shoot was reduced when plants were exposed to Fe deficiency (-26%) and bicarbonate (-40%). No significant differences were found in root FW (Fig. 2B). The plant dry weight (DW) showed the same trends (Fig. 2C).

40 a (a) 30 b cm 20 a a c 10 Figure 2. Shoot and root lenght (a), fresh b weight (b) and dry weight (c). The clear part 3 of the histogram corresponds to parameters a (b) g 2 b determined on the shoots; the dark area c indicates the parameters determined on the 1 roots. Three independent experiments in 0.3 triplicate (n=9) were performed. In the case a (c) g 0.2 of significant differences (P<0.05) values are b c marked with different letters. 0.1

+Fe -Fe +FeBic

2.3.3. Leaf chlorophyll determination 3 Fig. 3 shows the chlorophyll concentration a expressed on a FW basis. The total lack of Fe b

resulted in a larger decrease in leaf chlorophyll FW)

2 1 - c concentration with respect to the bicarbonate supply (-34% and -20%, respectively). g (mg

content 1 Figure 3. Change in the leaf chlorophyll concentration (mg g-1 FW) in plants grown for 9 days in control condition (+Fe), Fe deficiency (-Fe) and in the presence of bicarbonate (+FeBic). Chlorophyll Data are the means ± S.D. (n = 5). In the case of significant differences (P<0.05) values are marked with different letters. 0 +Fe -Fe +FeBic

2.3.4. Fe and P determination Table 2 reports the amount of apoplastic Fe in the roots from different growth conditions. The Fe trapped in the free space of roots grown in the presence of bicarbonate increased 4.9-fold, while in -Fe a decrease (-19%) was found. In the same plants, a decrease in Fe concentration was detected only in the tissues of –Fe roots, while at the shoot level the decrease was observed in both –Fe and +FeBic treatments (-70% and -53%, respectively). Concerning P concentration, a different trend was observed: while in the roots a slight decrease was found in absence of Fe and in the presence of bicarbonate (-17% and -26%, respectively), no significant difference was detected at the leaf level.

Table 2. Fe and P concentration ( mol g-1 DW) in leaves and roots of plants grown for 9 days in control condition (+Fe), iron deficiency (-Fe) and in the presence of bicarbonate (+FeBic). Data are means ± S.D. (n=5). In the case of significant differences (P<0.05) values are marked with different letters.

Fe P

shoot root simplast root apoplast shoot root

+Fe 4.44 0.23 a 1.13 0.09 a 0.80 0.03 b 503.14 32.25 a 518.41 41.44 a

-Fe 1.34 0.11 c 0.86 0.14 b 0.65 0.01 c 499.32 17.52 a 430.08 26.17 b

+FeBic 2.08 0.19 b 1.09 0.11 ab 3.91 0.25 a 449.95 29.54 a 384.94 13.97 b

2.3.5. Root Fe reduction and medium acidification in vivo In order to obtain a preliminary overview of the induction of Fe deficiency responses, the reduction and the acidification capacities were carried out in vivo by embedding the roots in agar (Fig. 4). The reduction activity was detected by using BPDS, which forms a stable red complex only with Fe2+ (Römheld, Müller and Marschner 1984), indicating that reduction of Fe3+ has occurred. Fig. 4A shows that plants grown in presence of Fe did not develop any colour, and conversely in –Fe treated plants a high reduction activity was present, mainly localized in the apical and sub-apical zones (Fig. 4B). Parietaria plants grown with bicarbonate showed the formation of a red colour less intense than –Fe treated plants (Fig. 4C). However, in this treatment the reduction activity appeared to be more diffuse along the root length. Medium acidification was determined in vivo by the change in the colour of the pH indicator bromocresol purple and it is shown in Fig. 4D-F. The pictures reported in the figure were taken approximately 2 h after agar embedding and show an acidification activity only in –Fe and +FeBic conditions (Fig. 4E and 4F). It is noteworthy the difference with the localization of the reduction activity, since the acidification appeared more diffuse in the –Fe treated

roots and more localized in the roots of bicarbonate-treated plants, particularly in the zone where the proteoid-like roots were present.

A B C

E F D

Figure 4. Visualization of Fe reduction (A, B, C) and acidification (D, E, F) activities of the whole root system. Roots were embedded in 0.75% agar medium and the reduction was 2+ determined, after about two hours, as the Fe -(BPDS)3 complex formation (red), whereas the acidification was detected as pH changes of the indicator bromocresol purple (yellow). From left: 9-day-old root grown under control condition (A, D), Fe deficiency (B, E), and +FeBic treatment (C, F). The experiment was repeated three times with similar results.

2.3.6. Fe reduction ability by phenolics Table 3 shows the results obtained testing the Fe reduction ability by both exuded and root accumulated phenolics. In particular, even if an activity was detected for all the root extracts, higher values were found for the +FeBic treatment. Regarding the exuded phenolics, an increase in Fe reduction ability was detected for both –Fe (+17%) and +FeBic (+32%) with respect to the control.

Table 3. Fe(III) reduction activity by phenolics extracted from Parietaria roots and plant exudates. Reduction is expressed as mol Fe mg-1 phenolics h-1. Data are means ± S.D. (n = 3). In the case of significant differences (P<0.05) values are marked with different letters.

Root extracts Plant exudates

Water-soluble Methanol-soluble

+Fe 0.10 0.01 b 0.15 0.02 b 122.12 6.54 c

-Fe 0.09 0.01 b 0.18 0.06 b 142.91 3.28 b

+FeBic 0.34 0.03 a 0.36 0.11 a 161.57 3.81 a

2.3.7. Strategy I response activities in plasma membrane preparations and root soluble extracts The assay of marker enzyme activities (vanadate-sensitive, nitrate- sensitive and azide-sensitive ATPases) carried out on plasma membrane preparations isolated from roots of differently treated plants indicate that the H+-ATPase activity was scarcely sensitive to nitrate and azide (less than 6% inhibition), while it was almost completely inhibited by vanadate (ca. 90%), indicating an enrichment in plasma membrane in the preparations used (Table 4). On these enriched preparations we have analyzed the Fe3+-chelate reductase and the H+-ATPase activities (Table 4). Both Fe-deficiency conditions induced an increase in FC-R activity in plasma membrane preparations from roots of Parietaria diffusa; in particular, in –Fe treatment the enhancement was approximately 2.3-fold with respect to control plants, while in the presence of bicarbonate the increase in FC-R activity was only approximately +51%. The H+-ATPase activity showed the same trend: both –Fe and +FeBic treatments showed an increased activity with respect to the control, but lower for the bicarbonate treatment (+95% and +45%, respectively).

Table 4. Enzymatic activities of typical Strategy I responses in root plasma membrane preparations and root soluble extracts from control (+Fe), Fe- deficient (-Fe) and bicarbonate (+FeBic) treated plants. For the H+-ATPase, the marker enzyme activities in plasma membrane-enriched fractions are reported. Data are the means ± S.D. (n=9). In the case of significant differences (P<0.05), values are marked with different letters.

Enzymes +Fe -Fe +FeBic

H+-ATPase 237.92 12.31 c 463.09 32.86 a 344.01 28.65 b nmol min-1mg-1 prot

Vanadate-sensitive 218.18 3.04 418.35 2.17 324.63 15.01

Nitrate-sensitive 14.30 2.33 24.45 1.92 21.70 3.15

Azide-sensitive 7.16 0.48 12.96 0.47 5.84 0.29

FC-R 189.96 16.21 c 435.59 22.17 a 287.03 16.04 b nmol min-1 mg-1 prot

PEPC 79.93 11.96 b 138.71 13.18 a 56.06 7.91 c nmol min-1 mg-1 prot

PEPC activity in root soluble extracts was differently affected in plants grown under Fe absence and bicarbonate supply conditions. In fact, an increase (+74%) in PEPC activity was observed only in -Fe, while in the presence of bicarbonate supply a slight decrease was induced (-30%) (Table 4). Western blot analysis revealed an increased level of the plasma membrane H+-ATPase protein in plants grown under both stress conditions. However, the increase was more evident under Fe-free nutrient solution than under bicarbonate supply, confirming the results obtained both in vivo and in vitro (cfr. Fig. 4 and Table 4).

Figure 5. Western blot analysis of H+-ATPase and PEPC carried out on root plasma membrane preparations and root soluble extracts from plants +Fe -Fe +FeBic grown for 9 days in control conditions (+Fe), Fe deficiency (-Fe) and in the presence of bicarbonate + (+FeBic), respectively. Plasma membrane or soluble H -ATPase proteins (15 g) were loaded on a discontinuous SDS- polyacrylamide gel (8%). After SDS-PAGE, proteins were electrophoretically transferred to PVDF membrane filters and two different antisera were used; one raised against the central domain of PM PEPC H+-ATPase of Arabidopsis thaliana (1:10 000) and the second one raised against a PEPC isoform of sorghum (1:1000). The experiment was repeated three times with the same results.

Polyclonal antibodies raised against sorghum PEPC were also used to assess the presence of this enzyme in root soluble extracts and to evaluate the expression level under stress conditions. The Western blot results are shown in Fig. 5. In the control lane the antibody reacted only against a polypeptide with an apparent molecular mass of 103 kDa. However, in Fe-deficient condition two polypeptides corresponding to 103 and 108 kDa, respectively, were detected, in agreement with previous studies (De Nisi and Zocchi 2000). No appreciable bands were evident in the lane corresponding to root soluble extracts grown under bicarbonate supply. These results are in good agreement with the activities presented in Table 4.

2.3.8. Shikimate pathway enzymes Since shikimic acid is a precursor in the biosynthesis of aromatic compounds, and consequently of phenolics, some enzymatic activities with a key role in the shikimate pathway were assayed (Fig. 6). In comparison with the control, Fe deficiency, especially when induced by bicarbonate, led to a significant increase in the activity of these enzymes. DAHPS, which is the key enzyme controlling the carbon flow towards phenolics metabolism, increased significantly when plants were

grown in the presence of bicarbonate (+33% with respect to the control). The same was true for SDH activity (+41%) in agreement with the fact that plants grown under bicarbonate were characterized by a higher phenolics content. The SK activity, which converts the SDH’s product in 3-phospho-shikimate, was affected in both Fe-deficient conditions (Fig. 6). The activity of EPSP synthase in extracts from –Fe roots did not significantly differ from that found in the control (Fig. 6). On the contrary, in presence of bicarbonate, the EPSP synthase activity increase about 3-fold with respect to the +Fe treatment. Finally, the PAL activity, the first enzyme in the phenylpropanoid biosynthetic pathway, was also increased significantly in both stress conditions, but more in +FeBic (+38% and +92%, respectively).

Erythrose-4-Phosphate + PEP 1.0 0.8 -1 DAHPS (Δ280 mg prot) a 0.6 b b 0.4 Figure 6. Enzyme activities of DAHP 0.2 shikimate pathway in extracts 80 of roots from control (+Fe), Fe- 3-Dehydroshikimate 60 b a deficient (-Fe) and bicarbonate b 40 (+FeBic) treated plants. Data SDH (nmol min-1 mg-1 prot) are the means ± S.D. (n= 9). In 20 300 a a the case of significant Shikimate differences (P<0.05) values are 200 b marked with different letters. SK (nmol min-1 mg-1 prot) 100 DAHPS, 3-deoxy-D-arabino- heptulosonate -7-phosphate a 120 Shikimate-3-Phosphate synthase ; SDH, shikimate dehydrogenase; SK, shikimate 80 b b EPSPS (nmol Pi mg-1 prot) kinase; EPSPS, 5-enol-pyruvyl- 40 shikimate -3-phosphate syntase; PAL, phenylalanine ammonia- 4 Phenylalanine a lyase. 3 b 2 c -1 -1 PAL (nmol min g FW) 1

Flavonoids p-Coumaroyl CoA Isoflavonoids

Stilbenes Anthocyanins Phenylpropanoid esters

2.3.9. Oxygen consumption

In order to obtain an overview of mitochondrial activity, the O2 consumption rate of apical root segments was determined. Fig. 7 shows that O2 consumption rate decreases in roots of Fe-deficient (-40%) with respect to those from Fe sufficient and bicarbonate treated plants. After

KCN addition, the total O2 consumption decreased by -17 % and -32% in control and +FeBic roots, respectively, whereas in the absence of Fe the decrease was larger, -57%. The presence of SHAM, which inhibits the alternative oxidase, caused a greater decrease in the consumption of O2 in control and +FeBic conditions (-91% and -94%, respectively), while in -Fe roots decrease was ca. 100% (Fig. 7).

2500 a IR KCN resistant KCN+SHAM resistant a a 2000 a

FW 1500 1

- b

g b

1 -

1000

min 2 c 500

nmol O nmol a a b

+Fe -Fe +FeBic +Fe -Fe +FeBic +Fe -Fe +FeBic

Figure 7. Oxygen consumption rate in excised apical root segments from plants grown for 9 days in control condition (+Fe), Fe deficiency (-Fe) and in the presence of bicarbonate (+FeBic). Data are the means ± S.D. (n = 5). In the case of significant differences (P<0.05) values are marked with different letters. IR, initial rate; SHAM, salycilichydroxamic acid.

2.4. Discussion A limited nutrient availability generally affects plant growth more than photosynthesis, enabling the allocation of photosyntates to the production of C-based secondary metabolites, such as phenolics (Leradau and Coley 2002). Accordingly, when root phenolics were quantified in Parietaria plants collected directly from the walls (Table 1), a correlation between their amount and the pH of the substrates was found. These data might suggest that plants respond to the pH variation through metabolic rearrangement, modifying the allocation of carbon skeletons between primary and secondary metabolism. Interestingly, in the same roots a simultaneous increase in the organic acid concentration occurs (Table 1); these compounds are reported to be accumulated and exuded not only under Fe deficiency, but also under other nutrient deficiencies (Lin et al. 2011; Widodo et al. 2010). Subsequently, to investigate growth and physiological behaviour particularly concerning Fe uptake, rooted cuttings of Parietaria were grown in hydroponic medium and submitted to direct (-Fe) or induced Fe deficiency in the presence of bicarbonate (+FeBic). This last condition would mimic the natural environment where plants grow showing a stable phenotype. In fact, while the absence of Fe resulted in a significant decrease in the chlorophyll concentration causing the classical symptoms of chlorosis, in the presence of bicarbonate chlorophyll was less affected, showing weak or no symptoms of chlorosis (cfr. Fig. 1 and Fig. 3). However, under the latter condition a more significant decrease in shoot growth was observed, probably as an adaptive strategy to maintain an adequate level of Fe (and chlorophyll, as well) in the tissues (Fig. 2 and Table 2), preserving the photosynthetic activity as it has already been suggested for pear plants (Donnini et al. 2009). At root level, the -Fe condition induced the proliferation of lateral roots (Moog et al. 1995; Dasgan et al. 2002; Jin et al. 2008), while the bicarbonate supply induced a shorter root system, with the appearance of structures similar to “proteoid roots” (Purnell 1960), maintaining the same root mass as the control plants. Proteoid roots, developing from secondary and tertiary lateral roots, provide an enhanced surface of contact between plant and soil which is useful for the release of nutrient-solubilising compounds and

for the uptake of nutrients from the rhizosphere (Dell’Orto et al. 2003; Lambers et al. 2003). The presence of such structures only in plants grown with bicarbonate suggests that this should not be a specific response to Fe deficiency but to a more general condition of low nutrient availability: indeed P deficiency has been found to be induced in calcareous soils as well (Guerinot Yi 1994). In 9-d-old Parietaria we found a slightly decrease in P concentration in both Fe deficiency conditions (Table 2). Preliminary findings obtained by testing Parietaria in the field prompted us to investigate both the accumulation and exudation of organic compounds. In particular, as suggested by Jin et al. (2007), phenolics could play an important role in the mobilization of Fe accumulated in the highly negatively charged cell wall, probably through chemical chelation and reduction operated by phenolics themselves. We found an increase in the accumulated and exuded phenolics in both conditions of Fe deficiency that was higher in the presence of bicarbonate. However, the release of phenolics into the exudates in these conditions, which has been attributed mainly to proteoid roots tissues (Weisskopf et al. 2006), appears to be scarce when compared with the amount accumulated in the roots (Table 1). Table 3 shows the capacity of these phenolics to reduce Fe(III)-EDTA in vitro, demonstrating that they could effectively influence the reduction and then the mobility of Fe-chelates both in the apoplast and in the external medium. The major Fe reduction efficiency detected under bicarbonate supply might be ascribed to relative abundance of specific compounds (results not shown), since the reducing capacity has been attributed to the presence of particular groups or double bonds in the molecular structure (Bors et al. 1990). The analysis of some key enzymes of the shikimate pathway shows a good correlation between the concentration of phenolics and an increase in the activities of several enzymes in their biosynthetic pathway. In fact, an enhanced amount of phenolic compounds, as well as increases in the activities of DAHP synthase, SDH, SK and PAL, was found in both Fe- deficient conditions, and the increase was larger under the bicarbonate treatment with the exception of SK (see Table 1 and Fig. 6). These results are also in agreement with data by Lan et al. (2011) who find a robust

increase in the amount of enzymes linked to the phenylpropanoid pathway induced by Fe deficiency. In the presence of bicarbonate, we found also an increase in the EPSP synthase, the sixth enzyme of the prechorismate pathway, whose over-expression has been associated to the tolerance to the herbicide glyphosate, which blocks the synthesis of aromatic compounds (Amrhein et al. 1983; Comai, Sen and Stalker 1983; Reinbothe, Nelles and Parthier 1991). Interestingly, resistance to glyphosate has been reported in Parietaria (Protopapadakis 1985). When organic acids are measured in both roots and exudates, a significant increase was detected in roots grown under both stress conditions. Under Fe, but also P deficiency, organic acid excretion would increase plant efficiency, through a decrease in the pH of the medium and consequently a solubilisation of Fe and P. Our data show that in the presence of bicarbonate, citric acid is the main exuded organic acid (Table 1), as already reported in other species (Massonneau et al. 2001; Jelali et al. 2010). On the other hand, it has been reported that malate is poor at mobilising micronutrients from the soil, whilst citrate is capable of mobilising significant quantities (Jones and Darrah 1994). Furthermore, Langlade et al. (2002) reported that the ratio of malate to citrate is higher only in the apex and juvenile proteoid roots. P. diffusa, a Strategy I plant species (Dell’Orto et al. 2003), responds to Fe deficiency by inducing the FC-R and H+-ATPase activities (Fig. 4B and 4E and Table 4), the last one attributable to an increase in the protein expression (Fig. 5). An increase in Strategy I enzymatic activities were also observed in +FeBic, although to a minor extent, which could be attributed to the presence of Fe in the medium, that might in part satisfy the plant requirements. Interestingly, the results obtained in vivo show that when roots are grown with bicarbonate, the reduction activity appears widely distributed along the roots (Fig. 4). We suggest that for this treatment, the FC-R activity measured in the plasmalemma fraction and known to be localized mainly at apical and sub apical root zones (see –Fe treatment) could be only a part of the total Fe reduction activity detected in vivo; the release of reductants (e.g. phenolics) could be responsible for the activity which appears to be present along the whole root length.

In the present work, an increase in the PEPC activity was observed only in –Fe; surprisingly, the bicarbonate supply in the presence of Fe induces a decrease in this activity and the protein expression in root soluble extracts shown by Western blot analysis confirms these results (Fig. 5). Another intriguing result obtained in Parietaria is the different presence of polypeptides reacting with the antibody. In fact, only in the absence of Fe we found both the 103 and 108 KDa as reported in the PEPC literature (see De Nisi and Zocchi 2000 and references therein). Phosphoenolpyruvate (PEP) is a substrate for the reactions catalyzed by both PEPC and DAHP synthase. We suggest that in the presence of bicarbonate, the PEP produced by glycolysis could be in part channelled into the shikimate pathway and converted into phenolics, which are abundant in this condition. In a mural habitat, the pH buffered to 7.2-8.3 causes precipitation of Fe along with other nutrients. In this condition the Strategy I activities might become inadequate to assure Fe supply if not by means of an extreme effort by H+-ATPase activity to counteract the buffering effect of bicarbonate. In this case, the Fe pool stored in the root apoplast could represent a valid extracellular reserve; the more a plant will be able to mobilize such pools, the more it will be competitive in these growing conditions. We show that P. diffusa responds to the presence of bicarbonate through an overproduction of phenolics and organic acids. Furthermore, data obtained through an experimental approach in hydroponic culture, suggest that this behaviour could be related to a metabolic shift involving particularly the PEP-consuming reactions. In the absence of Fe, Parietaria would need more energy for the activation of the Strategy I responses. However, in this condition, the primary source of energy (mithochondrion) is impaired (Fig. 7, see also Vigani, Maffi and Zocchi 2009). It has been shown that in Fe-deficient cucumber roots the rate of glycolysis and the PEPC activity are increased to produce the substrates useful to sustain FC-R and H+-ATPase activities (Espen et al. 2000; De Nisi and Zocchi 2000; Zocchi 2006). The PEP produced could then be addressed to the sustenance of biosynthesis of both phenolics and organic acids. In the presence of bicarbonate, Fe is present in the external and apoplastic solution even if in unavailable forms, and the

metabolic choice of Parietaria might be the activation of the shikimate pathway to produce phenolics, useful to Fe desorption from the cell wall. In the presence of Fe and bicarbonate, the mitochondrial electron transport chain is unaffected in comparison with the control (Fig. 7), remaining the primary mechanism to produce energy; furthermore, since the mitochondria are not significantly affected, the TCA cycle could better produce organic acids, explaining the increase in these compounds seen in this condition. The diversion from primary metabolism for a part of the PEP pool is in agreement with the decrease in the PEPC activity observed in the presence of Fe and bicarbonate. This enzyme is negatively regulated by L-malate (Chollet, Vidal and O’Leary 1996), whose concentration increases under this condition and, as proposed by a recent work (De María et al., 2006), by shikimate, which has been shown to exert a putative inhibitory effect on the PEPC, reinforcing the hypothesis. In conclusion, while Parietaria grown in the absence of Fe shows metabolic responses similar to other Strategy I plants growing in the same condition, under +FeBic this species is able to diversify its response by increasing alternative pathways for Fe solubilisation. In calcareous soils, where Fe is present but scarcely soluble and more likely “trapped” in the cell wall, the aptitude of the secondary metabolism to assume a relevant role could be responsible for the Parietaria’s exceptional efficiency. We summarize the results of this and previous studies (Dell’Orto et al. 2003) by proposing a model for metabolic rearrangement in Parietaria (Fig. 8).

+Fe

Fe Fe Fe

Fe H+ - + e H e- H+ e- e- Fe H+-ATPase Fe organic Fe ATP acid phenols IRT1 H+ Fe Fe PEPC e- pathway Shikimate NAD(P)H pathway Fe FCR e- PEP Fe glycolysis

+ TCA H e-

-Fe +FeBicc

org acid phenols H+ Fe H+ phenols H+ H+ HH++ - e- e e- H+

phenols

H+ H+-ATPase organic e- ATP phenols acid IRT1 Fe - Fe e PEPC Shikimate NAD(P)H pathway pathway FCR

phenols Fe PEP e- glycolysis phenols TCA e-

Figure 8. Schematic representation of metabolic changes occurring in roots of P. diffusa growing in control conditions (+Fe), Fe deficiency (-Fe) and in the presence of bicarbonate (+FeBic). Dotted lines indicate the metabolic pathways and Strategy I activities in the control condition (+Fe). In –Fe and +FeBic panels, the arrow thickness would indicate the change in the metabolic flux and Strategy I activities. FC-R, Fe3+-chelate reductase; IRT1, Iron Transporter 1; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase.

Aknowledgments We wish to thank Dr. M. Dell’Orto, Dr. G. Vigani and Dr. M. Wellens for continuous advice and for a critical reading of the manuscript. We thank Dr. G. Vigani for the schematic representation of Fig. 8. We wish also to thank Dr. R. Serrano (Universidad Politécnica, Valencia, Spain) and Dr. J.Vidal (Université de Paris-Sud) for providing the antybodies used in this work.

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CHAPTER 3 ______

Comparison of different alkaline pH growth conditions (bicarbonate and organic buffer media) on Parietaria judaica in a time course analysis. 70

ABSTRACT Fe deficiency-induced chlorosis, otherwise known as lime-chlorosis, is a major nutritional disorder in crops growing in calcareous soils that lead to huge losses in terms of quantity and quality of products. Calcareous soils represent 30% of the Earth land surface and are characterized by high pH values. The high alkalinity of calcareous soils causes a dramatic decrease in Fe bioavailability. Nevertheless, many wild species are well adapted to live (i.e. calcicole species) in calcareous environments overtaking the low bioavailability of Fe. The adaptation of a species most frequently depends on more than a single trait as natural selection operate on the whole organisms. Hence the set of traits shaped by natural selection in wild species represent a successful strategy to live in a given environment, thus identify the main functional traits involved in tolerance to Fe low availability of a wild species could offer interesting inputs for breeding programs in agriculture. Parietaria judaica is a wild sinantropic plant that grows successfully in highly calcareous environments without showing any symptom of chlorosis. As a dicot, P. judaica encompasses all the inducible metabolic changes of a Strategy I plant at biochemical, physiological and morphological level. Moreover, other non-specific responses are induced in P. judaica, such as tissue accumulation and root exudation of organic acids and phenolic compounds. Probably these non-specific responses confer to the high efficiency demonstrated in the acquisition of Fe. In the present work, in a 7 day time course, the root accumulation and exudation of the main low molecular weight organic acids and phenolic compounds was analysed in P. judaica grown in hydroponics under direct or induced Fe deficiency conditions. Two different alkaline conditions were applied in order to distinguish the effect due to the high pH from the responses induced by the presence of high concentrations of calcium carbonate. It was also analysed the activity of two key enzymes of primary metabolism - PEPC and G6PDH. The great efficiency exhibited by P. Judaica in high calcareous environments suggested high metabolic flexibility that enables to shift the metabolic fluxes in order to adapt the plant’s physiology to environmental conditions. 71

3.1. Introduction In plants iron (Fe) is required for a wide range of biological functions like photosynthesis, respiration and chlorophyll biosynthesis. Notwithstanding Fe is the second most abundant metal in the Earth’s crust it is not readily available for plants due to its low solubility. Actually, in well aerated soils at pH 7.0 Fe3+ and Fe2+ concentrations are less than 10-15 M, a level far below that required for plants for optimal growth (Marschner, 1995; Kim and Guerinot, 2007). Furthermore, in well aerated soils Fe occurs principally in oxidized forms - Fe(III) - that cannot be taken up by most plants, which can acquire just Fe(II). Accordingly, plants must be able to render these elements available in order to survive and compete successfully. Dicots and monocots plants (except Poaceae) have developed active metabolic adaptations to cope with low Fe availability, known as Strategy I. In Strategy I, a ferric chelate reductase (FCR) located at the root cell plasma membrane converts FeIII-chelates to Fe(II), and a Fe regulated transporter (IRT1) moves the ferrous ion across the plasma membrane into the cell. Moreover, in various species a H+ pumping activity, carried out by the plasma membrane H+-ATPase, is increased under Fe deficiency. The extrusion of H+ outward the cell acidifies the apoplast and the rhizosphere incrementing Fe solubility and generating an electrochemical gradient useful to drive the cation uptake (Rabotti and Zocchi, 1994; Dell’Orto et al., 2000; Santi et al., 2005; Santi and Schmidt, 2008, 2009). The activation of the Strategy I mechanism enhances the request of ATP and NAD(P)H+ for H+ extrusion and reduction processes, respectively, resulting in a general metabolic rearrangement. Among the metabolic changes that occur under Fe starvation, an increased activity of many glycolytic and TCA enzymes and of PEPCase was found, indicating a rising in carbohydrate catabolism (Vigani et al. 2011). Fe deficiency induces also an accumulation of low molecular weight organic acids (LOAs), mainly malate and citrate, in roots, xylem sap, leaf apoplastic fluid and whole leaves. Organic acid accumulation in Fe-deficient plants can improve long-distance Fe transport (López-Millán et al. 2000; López- Millán et al. 2001), and since the production of organic acids is protogenic, it could also contribute to the control of cytosolic pH and 72 feed the increased activity of the plasma membrane (PM) H+-ATPase (Zocchi, 2006). In addition, many works have shown increased root exudation of a variety of organic compound as a response to Fe deficiency, to increase the ferric ion solubility and/or support the reducing capacity of Fe(III) on the root surface (Dakora and Phillips, 2002; Tomasi et al., 2008; Cesco et al., 2010). Phenolic compounds have been reported as the main components in root exudates in Fe deficiency conditions and it has been suggested their implication in Fe uptake (Jin et al., 2007). It is well known that the presence of carbonates in soils cause a decrease in the solubility of Fe and other micronutrients reducing their exchangeable capacity with plants (Lindsay and Schwab, 1982; Boxma, 1972; Loeppert, 1986). In fact, at high soil pH there are several essential elements, notably phosphate, Fe and manganese (Mn) that are scarcely present in soluble or easily available form. Fe deficiency-induced chlorosis, otherwise known as lime-chlorosis, is a major nutritional disorder in crops growing in calcareous soils. Calcareous soils represent 30% of the Earth land surface, are characterized by high pH values and may contain high concentrations of HCO3- ions in the soil solution. On one hand alkaline pH dramatically reduces Fe solubility, but on the other it has been suggested that the presence of HCO3- interferes with the Fe uptake physiological processes. It was found higher Fe content in roots of plants grown under calcareous condition respect to those grown on neutral and non-calcareous conditions, indicating that low Fe availability in soils is not a problem per se, but rather a question of Fe uptake from the apoplast (Mengel, 1994). In the soil solution HCO3- certainly acts buffering the rhizosphere and the root apoplast thus neutralizing the H+ extruded by H+-ATPase. Furthermore, some studies have shown that alkaline pH and especially high HCO3-concentrations strongly inhibit the activity of the FCR which is a necessary step in Fe acquisition of Strategy I plants (Kosegarten et al., 2004). A study conducted on graminaceous species (sorghum, barley and maize) grown at different concentrations of HCO3- suggested that the impaired Fe acquisition observed at high concentration of HCO3- is attributable to the root growth inhibition (Alhendawi et al., 1997). Studies conducted on wild plants grown on 73 calcareous soils have suggested that chlorosis is caused by the immobilization of Fe in non-active forms rather than by a decrease of its total concentration in leaves or roots (Zohlen and Tyler, 2000; Zohlen, 2002). Lucena et al. (2007) working on Arabidopsis and other dicotyledonous crop species (pea, tomato and cucumber) have suggested that high contents of HCO3- could cause Fe deficiency symptoms by inhibiting the expression of several Fe acquisition genes. The exact mechanism by which HCO3- acts was not yet completely elucidated. Anyway, many plants species, despite the low concentration and availability of Fe in calcareous soils, can develop successfully without showing signs of chlorosis. Parietaria judaica is a wild sinantropic plant that grows successfully in high calcareous environments such as walls and is highly Fe-efficient.

Figure 1.Parietariajudaica grown in a rock crack.

Just few works have been done on P. judaica regarding its adaptation to limestone. A previous study has confirmed that P. judaica exhibits the typical Strategy I response to Fe deficiency: increased FCR activity; high capacity to acidify the rhizosphere through the extrusion of H+. Anatomical and morphological changes have also been observed in response to low Fe availability. P. judaica grown under low Fe availability develops proliferation of secondary roots and a “proteoid” root like feature, which are typical modifications induced in some Strategy I plants (Dell’Orto et al. 2003). Other non-specific responses of P. judaica to Fe deficiency are the accumulation and exudation of sugars, amino acids and 74 phenols.It seems that the high Fe efficiency of P. judaica lies on secondary mechanisms such as morphological modifications and exudation (Dell’Orto et al. 2003). Donnini et al. (2012) found an enhanced activity of enzymes directly involved in the polyphenols biosynthesis pathway (SDH and PAL); a significant increase in low molecular weight organic acids (citric and malic) both in root tissues and exudates when plants were grown under the presence of bicarbonate. In Fe-starved conditions a common metabolic response concerns the increase of PEPC activity that plays a key role both in the pH stat control and in maintaining a high rate of glycolysis (Vigani and Zocchi, 2009). G6PDH catalyzes the first step in the pentose phosphate pathway that, among other roles, provideserythose-4 phosphate (E4P), one of the substrates required by the shikimate pathway, which in turn leads to the synthesis of phenols. Furthermore, G6PDH has been suggested to be involved in the cell redox homeostasis (Singh et al. 2012).

In the present work the responses of P. judaica grown in hydroponics under direct and induced Fe deficiency conditions were analyzed. In particular two high alkaline treatments were set up: one containing

CaCO3 and NaHCO3 having a pH value of 8.3 in order to mimic a calcareous soil condition and another one buffered with Tricine at a pH 8.3. These alkaline treatments were carried out in order to separate the - 2+ effects due to a calcareous medium (presence of HCO3 and Ca in solution) from those of the high pH on Fe acquisition. Determination of citric and malic acid and phenolic compound content in roots and exudates was conducted in a time course, as well as the assay of PEPCase and G6PDH activities.

3.2. Materials and methods 3.2.1. Plant material Cuttings of P. judaica were taken from a mother plant and transplanted in an aerated half nutrient solution for 10 days. Rooted plant cuttings were then transferred to 10 L plastic tanks (40 plants per tank) with four different hydroponic conditions: +Fe (control, full nutrient solution adjusted to pH 6.2), -Fe (full nutrient solution in the total absence of Fe, 75 brought to pH 6.2), +Bic (full nutrient solution with addition of 0.5 gL-1

CaCO3 and 15 mM NaHCO3 which brought the pH to 8.3), Trc (full nutrient solution buffered with Tricine and adjusted to pH 8.3). Where required pH was adjusted with NaOH. The treatment buffered with Tricine was introduced to distinguish the low availability of Fe due just to high pH from the effect of the presence of bicarbonate on Fe availability. Treatments were carried out for 7 days in a growth chamber under 16/18 h light/dark regime with cool-white light 200 mol m-1 s-1, 27/21°C temperature range, 65-75% relative humidity. Plant samples were collected at first, third, fifth and seventh day of the overall treatment.

3.2.2. Collection of root exudates Fifteen 7-d-old plants for each treatment were transferred to 250 mL distilled water with 10% (v/v) Micropur (Katadyn, Minneapolis, MN) to prevent microbial activity. Root exudates were collected for a period of 24 h under continuous aeration. The exudate solution collected for the total phenolic compound determination was acidified to pH 3.5-4.0 with HCl before being freeze dried. For all the other measurements the exudate solution was freeze dried without adjusting the pH. The lyophilized material was resuspended in 3 mL distilled water and filtered through 0.45 m Millipore Millex-HN.

3.2.3. Enzymatic determination of organic acids Root samples were collected, carefully rinsed in distilled water and homogenized in 10% (v/v) perchloric acid following a 1:1 ratio g/mL and centrifuged at 10,000 g for 15 min. The pH was brought to 7.5 with 0.5 M

K2CO3 to neutralize the acidity and to precipitate the perchlorate. Extracts were successively centrifuged at 15,000 g for 15 min and the supernatant was recovered. Citric and malic acid concentration in both root extracts and exudates was determined enzymatically as follows: Citric acid was assayed by coupling its conversion to oxaloacetate by citrate lyase (CL, EC 4.1.3.6). In the presence of malate dehydrogenase (MDH, EC 1.1.1.37) and lactate dehydrogenase (LDH, EC 1.1.1.27), the 76 oxaloacetate and its decarboxylation derivative, pyruvate, are reduced to L-malate and L-lactate by reduced nicotinamide adenine dinucelotide (NADH). The amount of NADH oxidized to NAD+ in these reactions is proportional to the amount of citrate present. Malic acid was analysed by the enzymatic conversion of L(−)malate to oxaloacetate in the presence of NAD+ and L(−)malate dehydrogenase (MDH). In order to favour the reaction towards the production of NADH the enzyme glutamate-oxaloacetate transaminase (GOT, EC 2.6.1.1) is used. Variations in NAD/NADH+ amounts were detected by monitoring the absorbance changes at 340 nm and were performed at 26°C and in a final volume of 1 mL. At least three independent assays were performed for each treatment.

3.2.4. Determination of phenolic content Phenolic content in root tissues was determined in two different extraction media: 100% distilled water and 100% methanol. Root samples from the different treatments were homogenized in the extraction medium with a volume ratio 1:1 (w/v). The homogenates were centrifuged at 10,000g for 15 min and the supernatants collected. The amount of phenolics in root extracts and exudates was determined spectrophotometrically at 750 nm with the Folin-Ciocalteau reagent using gallic acid as a standard (Donnini et al., 2012). Three replicates of three independent experiments were performed for each treatment (n=9).

3.2.5. Determination of apoplastic Fe Apoplastic Fe was determined in a time course at 1,3, 5 and 7 days. Roots from 5 plants per treatment were transferred to a beaker with 0.5 mM

CaSO4 under vigorous aeration. After 10-15 min, roots were transferred to

40-ml tubes with 21 ml of 10 mM MES, 0.5 mMCa (NO3)2, 1.5 mM 2,2’-bipyridyl (pH 5.5) at 25°C. Tubes were covered with a cotton plug and N2 was bubbled through the solution. After 5 min, 1 mL of 250 mM

Na2S2O4 was added. The A520 of the solution (A520 of 1 mM 77

Fe[bipyridyl]3 = 8.650) was determined on 2 mL aliquots as reported by Bienfait et al. (1985). The aliquots employed for the determinations were returned to the tube and left for 1h in the dark. The determination was carried out every 1.30 h until a constant value was obtained.

3.2.6. Soluble enzyme extraction and assay of PEPCase and G6PDH Roots sampled from different treatments were cut, rinsed in distilled water, and homogenated at 2-4°C in a buffer solution (1:1 mL/g) in the presence of 20% (w/w) PVPP. The buffer solution contained: 50mM Tris-

HCl pH 7.5, 10mM MgCl2, 10% glycerol, 1mM EDTA, 14 mM mercaptoethanol, 1mM PMSF and 10 mg mL-1leupeptin. The homogenates were filtered through 4 layers of gauze and centrifuged at 13,000g at 4°C for 15 min. Supernatants were centrifuged at 100,000g at 4°C for 30 min. The final supernatants were collected and dialyzed for 3h at 4°C against 2 L of leupeptine-free buffer solution. The dialyzed extracts were used for enzymatic assays of Phosphoenolpyruvate carboxylase (PEPC) and Glucose-6-phosphate dehydrogenase (G6PDH). PEPC (EC.4.1.1.31) activity was determined as in De Nisi and Zocchi (2000). The reaction was started by adding an aliquot of soluble enzyme extract. G6PDH (EC 1.1.1.49) was assayed according to Rabotti et al. (1995). The reaction was started by adding an aliquot of soluble extract.

3.2.7. Statistical analysis Values are expressed as means SD. One way ANOVA analysis was used to test any statistical difference among data. Differences at P<0.05 were considered to be significant. Statistical differences have been expressed by different letters. All statistical analyses were carried out with SPSS v.15.0.1 (SPSS Inc.)

3.3. Results

3.3.1. Malic and citric acid content in root tissues and exudates Table 1 shows the contents of LOAs in roots at 1, 3, 5 and 7 days of treatment.

-1 A Malic acid content in roots ( g g FW)

+Fe -Fe Bic Tric

T1 264.66 20.32 c 177.49 12.67 e 452.24 47.58 a 289.34 20.75 c

T2 221.88 19.23 cd 150.78 11.73 e 400.81 35.51 b 259.01 17.37 d

T3 111.17 9.69 ef 157.98 13.62 ef 348.65 32.57 c 241.79 23.78 d

T4 105.89 8.45 f 167.76 18.84 e 307.05 20.62 c 148.70 10.94 e

-1 B Citric acid content in roots ( g g FW)

+Fe -Fe Bic Tric

T1 748.11 70.27 b 470.55 35.87 d 611.63 54.90 bc 682.76 45.20 bc

T2 183.03 15.98 f 377.66 35.76 e 764.84 74.38 b 787.29 70.26 b

T3 90.89 10.34 f 452.72 46.72 de 1148.72 102.39 a 963.80 82.58 ab

T4 101.23 9.56 f 525.34 49.84 d 1346.56 128.83 a 1024.35 99.87 b

Table 1.Time course of the amount of LOAs in root tissues in +Fe, -Fe, Bic and Tric treatments. A. content of malic acid; B. content of citric acid. T1, T2, T3 and T4 correspond to sampling at 1, 3, 5 and 7 days. Data are the means ± SD (n=7). Statistical differences (P< 0,05)are indicated with different letters.

Regarding malic acid content (Table 1 A), values gradually diminished during the treatment in +Fe, Bic and Tric condition). The highest decreases were registered for +Fe (control) and Tric, in which nearly 60% and 50% decrease was registered, respectively, comparing with their initial amounts at the beginning of the treatment. In bicarbonate 79 condition (Bic) a 32% decrease was recorded with respect to the initial content at the beginning of the treatment. In –Fe condition the absolute values did not present significant differences.

Malic and citric acid content at 7 d a

c g/g g/g f.w. a b c d b a

Malic ac. Citric ac. Figure 1. Comparison of malic and citric acid root contents expressed as g g-1FW at the end of the treatment for +Fe, -Fe, Bic and Tric treatments. Error bars indicate standard deviation (n=7). Different letters indicate significant difference (P<0.05). Significant differences were calculated for each individual compound and are indicated in different colours.

Citric acid content in roots shows a different trend. While in control condition citrate diminished by 86% after 24 h (T1) remaining then constant, the other treatments showed a significant increase. Bic and Tric conditions exhibit the highest accumulation of citric acid (2 and 1.5 fold respectively) at the end of the treatment compared with their contents at T1. Figure 1 compares the malate and citrate content at the end of the experiment. All stressed treatments (-Fe, Bic, Tric) show an increase of malic and citric contents with respect to the control (+Fe). The accumulation of malic and citric acids is considerably higher in the presence of bicarbonate (Bic), 3-fold and 13-fold, respectively, compared to the control (Fig. 1). In Fe starved roots (-Fe), malate accumulation increases by 60% respect to the control. Both alkaline treatments increase citric acid 13-fold and 10-fold,respectively, compared with the control. In 80 direct Fe deficiency condition (-Fe) citrate accumulate by 5 fold respect to the control (Fig. 1). Table 2 A and B shows the concentrations of malic and citric acid in exudates collected for 24 h from roots of plants submitted to the Fe- deficient treatments for 1, 3, 5 and 7 days.

A Malic acid in root exudates ( g g-1FW d-1)

+Fe -Fe Bic Tric

T1 n.d. n.d. n.d. n.d.

T2 n.d 0.96 0.12 de n.d. n.d.

T3 0.87 0.10 e 6.05 0.74 ab 3.47 0.51 c 1.22 0.25 d

T4 1.68 0.28 d 7.67 0.88 a 5.17 0.65 b 2.83 0.24 c

B Citric acid in root exudates ( g g-1 FW d-1)

+Fe -Fe Bic Tric

T1 n.d.* 0.53 0.10 f n.d. 0.42 0.09 f

T2 n.d 1.15 0.21 e 1.96 0.27 d 1.06 0.12 e

T3 n.d 4.73 0.50 b 3.47 0.35 c 2.27 0.25 d

T4 0.33 0.04 f 5.89 0.49 b 10.87 0.84 a 5.96 0.37 b

* n.d. no detectable

Table 2.Time course of the amount of LOAs in root exudates in +Fe, -Fe, Bic and Tric treatments. A. malic acid; B. citric acid. T1, T2, T3 and T4 correspond to sampling at 1, 3, 5 and 7 days. Data are the means ± SD (n=7). Statistical differences (P< 0,05)are indicated with different letters.

Malic acid content in exudates increases in all treatments respect to the control. Malic acid was detected since T3 sampling for almost all the treatments. The highest increases in malic concentration were recorded for -Fe and Bic (3-4 fold at T4). –Fe roots respond first with respect to the other treatments exuding malic acid at T2, increasing consistently at T3 while the successive amounts at T4 and T5 remain constant. 81

Similarly, citric acid content in exudates exhibits a strong increase in all treatments. Bic condition increased 33-fold while –Fe and Tric showed an 18-fold increment with respect to the control. In general, citrate could be detected earlier in exudates in comparison to malate. In –Fe and Tric conditions citric acid was found in exudates after 24 h of treatment (T1).

3.3.2. Phenolic compounds in root extracts and exudates Table 3 A and B shows the results concerning the total phenolics measured in root water and methanol extracts. Water soluble contents of phenolics are quite variable along the time course and no significant correlation could be found between concentration and time for any of the treatments. At the end of the treatment (T4) direct and induced Fe deficiency treatments exhibit a higher accumulation of water soluble phenolics respect to the control. Bicarbonate (Bic) condition induces an accumulation of phenolics of about +75% compared with the control while –Fe and Tric treatments increase by 40% their phenolic content without any significant difference between them. The methanol soluble phenolics showed a positive correlation between concentration and time only in Bic condition, in which the accumulation of phenolics exhibits a 2-fold increase at the end of the treatment. Fe starved treatment showed no variations of contents in the time course, while the control condition decreased its content in phenolics, slightly less than 50%, at the end of the treatment. Phenolic compounds determined in methanol extracts showed a consistent increase in all stressed conditions at final sampling (T4) when compared with control. Bicarbonate treatment accumulated 4-fold more phenolics than control while –Fe and Tric increased 2-fold. Similarly to what registered for water extracts, Fe starved and Tric roots exhibit similar phenolic contents. The content of phenolics in root exudates collected in +Fe, -Fe, Bic and Tric is shown in Table 4.

A Water soluble phenolic compounds in roots (mg g-1FW)

+Fe -Fe Bic Tric

T1 1.09 0.08.f 2.076 0.12 ab 1.24 0.07 e 2.15 0.11 ab

T2 1.64 0.09 d 1.93 0.08 bc 1.65 0.10 d 1.84 0.07 c

T3 1.08 0.10 f 2.23 0.14 a 1.31 0.09 e 0.94 0.10 f

T4 0.97 0.09 f 1.37 0.11 e 1.71 0.12 cd 1.36 0.10 e

B Methanol soluble phenolic compounds in roots (mg g-1FW)

+Fe -Fe Bic Tric

T1 1.13 0.11 cd 1.28 0.12 c 1.05 0.12 cd 1.16 0.08 cd

T2 1.20 0.13 cd 1.13 0.11cd 1.50 0.10b 1.22 0.08cd

T3 0.74 0.06 e 1.02 0.09 d 1.22 0.09 c 0.66 0.05 e

T4 0.60 0.07 e 1.08 0.01d 2.21 0.13 a 1.09 0.99 d

Table 3. Content of phenolic compounds in roots of P. judaica along a time course for +Fe, -Fe, Bic and Tric treatments. A. concentration in root extracts with water; B. concentration in roots extracts with methanol. Values are expressed as the means standard deviations. Different letters indicate the significant differences (P=0.05) among values.

Total phenolic compounds in root exudates (mg g-1FW d-1)

+Fe -Fe Bic Tric

T1 0.021 0.001 f 0.113 0.012 d 0.052 0.007 e 0.140 0.025 c

T2 0.020 0.002 f 0.213 0.021 c 0.093 0.004 d 0.124 0.081 c

T3 0.031 0.001 f 0.272 0.022 b 0.112 0.014 c 0.107 0.023 c

T4 0.030 0.002 f 0.314 0.026 a 0.126 0.011 c 0.115 0.012 c

Table 4. Time course of total phenolics in exudates from control (+Fe), Fe starved (-Fe), bicarbonate (Bic) and alkaline organic buffer (Tric) treated plants. Amounts are averages coupled with standard deviation. Different letters indicate the significant differences (P=0.05) among values (n=9).

The results obtained are one order of magnitude higher respect to a previous work (Chap. 2, par. 2.3.1. Table 1).In fact, the method for collecting exudates has been modified in the present study. Phenolics are more stable at low pH and precipitate at high pH so, to avoid loss of material and to maintain the structure of compounds, the collected solutions were acidified before being freeze dried. Phenolic compounds have been detected in exudates at the first sampling date (T1) in all treatments. At T1 higher amounts of phenolics were found in all stress treatments respect to the control; -Fe and Tric show 5 fold and 7 fold increases, respectively.Exudates from –Fe and Bic conditions showed a gradual increase in phenolic content over the time course, reaching a maximum level at the end of the treatment which resulted 3- and 24-fold higher with respect to the beginning of the treatment. In +Fe and Tric conditions the concentrations remained constant along the treatment. All stress treatments exhibit a higher content of phenolics with respect to the control at the end of the treatment (T4). The highest content of phenolics was found in -Fe exudates that exhibit a 11-fold increase with respect to the control at the end of the treatment. Bic and Tric treatment increased 4-fold respect to the control at the end of the treatment and showed no significant differences in the phenolic content of exudates between them.

3.3.3. Root apoplastic Fe The results of root apoplastic Fe content obtained along the time course for +Fe, -Fe, Bic and Tric treatments are shown in Table 5. The initial amount of apoplastic Fe of rooted plants was determined (T0) before imposing the treatments (1.41 0.17 mol g-1FW). In the control condition after 1 d of treatment a surprisingly 2-fold accumulation of Fe was registered, than rapidly decreased reaching a value comparable with that found at T0. In Fe starved conditions the content of Fe in the apoplast decreased until reaching zero at T3 (5 d). In agreement with previous results (cfr. Chapter 2, par.2.3.4.), the Bic condition exhibited a strong accumulation of Fe, reaching a 2.7-fold increase at T3 respect to 84

T0 and remaining almost constant until the end of the treatment (T4). In Tric condition an increase of 23% was recorded at the end of the treatment. No significant difference was found after 7 d (T4) between control and Tric condition, while in bicarbonate the accumulation was of 4-fold with respect to the control at the end of the treatment.

Apoplastic Fe content ( mol g-1FW)

+Fe -Fe Bic Tric

T1 3.04 0.25 b 0.98 0.15 d 1.30 0.15 c 0.90 0.10 d

T2 1.39 0.23 c 0.42 0.24 f 2.85 0.24 b 0.61 0.052 e

T3 1.04 0.12 cd 0.03 0.002 g 3.86 0.37 a 0.62 0.063 e

T4 0.94 0.05 cd 0.04 0.006 g 3.45 0.41 ab 1.11 0.19 g

Table 5.ApoplasticFe concentration ( mol g-1 DW) in roots of plants grown for 1, 3, 5, 7 days in control condition (+Fe), iron deficiency (-Fe), in the presence of bicarbonate (+FeBic) and alkaline organic buffer (Tric) treated plants. Data are means ± S.D. (n=5). In the case of significant differences (P<0.05) values are marked with different letters.

3.3.4. PEPCase and G6PDH activity PEPCase is considered a key enzyme under Fe deficiency conditions, both in terms of pH stat regulation and as a driving force for glycolysis (De Nisi and Zocchi, 2000). PEPCase activity showed a general and gradual decrease in all treatments (Tab. 6A) during the time course: the decrease was sharper in Tric condition, leading to a low enzymatic activity at the end of the treatment. In -Fe condition the PEPCase activity decreased at the beginning of the treatment, then reaching a constant value. Table 6B shows the results of the time course for the G6PDH activity. In control condition G6PDH activity at the end of the time course was about 21-fold higher respect to the activity at the beginning of the treatment. In –Fe and Bic conditions it decresed by 8- and 6-fold, respectively, in relation to their initial activity rates.

A PEPCase activity (nmol min-1mg-1prot.)

+Fe -Fe Bic Tric

T1 126.07 10.90 bc 118.74 10.56 bc 168.86 15.20 a 143.85 11.73 ab

T2 96.61 8.25 c 94.02 9.34 c 129.46 13.00 b 117.57 10.94 b

T3 67.24 6.84 d 82.68 6.79 c 70.00 5.24 cd 78.79 6.51 c

T4 62.54 4.70 d 90.80 8.90 c 83.77 5.77 c 24.61 3.70 e

B G6PDH activity (nmol min-1mg-1prot.)

+Fe -Fe Bic Tric

T1 2.64 0.30 h 4.07 0.37 g 3.70 0.30 g 7.26 0.75 f

T2 35.82 2.96 b 32.03 2.74 b 2.48 0.32 h 9.46 0.89 e

T3 57.68 4.28 a 10.00 0.97 e 19.20 2.13 d 5.49 0.54 g

T4 56.00 4.30 a 6.23 0.75 f 24.00 2.36 c 7.65 0.81 f

Table 6. Enzymatic activities measured in dialysed root soluble extracts from +Fe, -Fe, Bic and Tric treated plants. A. PEPC (phosphoenolpyruvate carboxylase) and, B. G6PDH (glucose 6-phosphate dehydrogenase). Data are expressed as the means ± SD (n=9). Enzyme activity is expressed as nmol min-1mg-1prot. Significant differences are labelled with different letters (P<0.05).

In Tric condition G6PDH activity did not significantly change during the treatment. Comparing this enzyme activity at 7 d (T4) among treatments it can be observed that in direct (-Fe) and induced Fe deficiency (Bic, Tric) conditions G6PDH activity decreses by 9-, 2- and 7-fold, respectively.

3.4. Discussion The levels of LOAs in plants vary greatly among different tissues and different species. However, increase in their content and exudation by 86 roots has been reported by several works as a response to environmental stresses and in particular to nutritional constrains such as Fe deficiency (Abadía et al. 2002; Jelali et al. 2010; Rellán-Alvarez et al. 2011; Jimenez et al. 2011). In many Fe-efficient genotypes of different crop species, Fe uptake efficiency has been associated to the ability to accumulate organic acids, mainly malic and citric acid (Lopez Bucio et al. 2000). Different functions of organic acids could explain their accumulation in Fe deficient roots: control of cytoplasmic pH by the pH Stat mechanism (Reid and Smith, 2002); control of apoplastic pH (López-Bucio et al. 2000); acting as chelating agent of Fe in apoplast and rhizosphere; acting as chelating agent of Ca to solubilize P in the soil (Tyler and Strom, 1995) and to capture excessive Ca inside the cell (Bush et al. 1995); Fe plant translocation through the formation of different species of FeIII-citrate (Rellán-Álvarez et al. 2011). In agreement with previous works (Zocchi, 2006; López-Millan et al. 2009 and references therein), a strong accumulation of malic and citric acids was recorded at the end of the treatment in roots and root exudates of P. judaica subject to direct or induced Fe deficiency. In both alkaline conditions (Bic and Tric) P. judaica performs a clear response of accumulation of citrate. During the time course plants grown in the presence of an adequate supply of Fe (+Fe) exhibited a consistent decrease in malate root content. These data are well correlated with the decreasing activity of PEPC observed along the treatment. Also in the presence of bicarbonate (Bic) and tricine (Tric) the trend for malic acid content was correlated with the relative PEPCase activity. A previous study (Chapter 2, par. 2.3.7.) has shown an increased H+-ATPase activity in P. judaica roots grown in the absence of Fe, suggesting the need for a cytosolic pH-Stat mechanism. In fact, in the present work, Fe deficient roots exhibited increased PEPC activity respect to the control since the T3 sampling, and accordingly, a strong accumulation of malate occurs. An even higher accumulation of malic and citric acids (fig. 1) occurred in plants grown in the presence of bicarbonate, probably due to both the enhanced specific PEPC activity - and the high availability of the substrate HCO3 which needs to be removed. Actually, in a previous work (Donnini et al. 2012) a low PEPC activity was reported in bicarbonate condition but the assay was carried 87 out in non-dialyzed extracts. In the present work the enzyme activity assays were conducted on dialyzed root extracts in order to reduce the high malic acid concentration, which is known to inhibit the PEPC activity (De Nisi and Zocchi, 2000). Furthermore, in Bic condition the growth rate is significantly lower with respect to control, -Fe and Tric conditions (data not show). Under active growth conditions TCA provides many precursors of anabolic metabolism such as amino acids, so the replenishment of TCA becomes necessary and it could occur through the contribution of cytosolic malate. The lower growth rate in Bic–treated plants could result in a lower consumption of malate. In Tric condition, growing rates are similar to the control and the organic acids levels in roots and exudates are compatible with a low Fe availability (tab.1). In these plants probably there is no necessity to exert a strong control on cytosolic pH, in addition mitochondrial activity should not be impaired so organic acids could be supplied by TCA. It has been well documented that roots exude a variety of organic compounds that play multiple functions in plant nutrient acquisition. An increase in organic acid root exudation has been reported in several dicotyledonous species under Fe deficiency and their involvement in mobilization of Fe has been documented as well (Farrar and Jones, 2000). Some studies have pointed out the enhanced rate of exudation of citric, malic and oxalic acids in calcicole species with respect to calcifuges ones and ascribe their efficient acquisition of P and Fe to this ability (Ström et al. 2005). P. judaica greatly increase the malic and citric acid contents in exudates when subject to direct or induced Fe deficiency (tab. 2A and B). Both malate and citrate once exuded may undergo complexation with target metals as Fe(III), improving its low solubilisation particularly in calcareous soils (López-Bucio et al. 2000, Abadía et al. 2011). Citrate demonstrates better metal extraction capabilities than malate in alkaline medium. Citric acid shows a higher concentration than malate in exudates produced by P. judaica under alkaline conditions (Bic and Tric), while in direct Fe deficiency (-Fe) malate presents a higher amount than citric acid. In –Fe and Tric conditions small quantities of citric acid were 88 detected after 24 h (T1) of treatment (tab. 2A and B) suggesting the predominant role of citric acid in the response to Fe deficiency. In calcareous soils the alkaline pH decreases the availability of many nutrients other than Fe, in particular P constitutes the main limitation since it precipitates as Ca3(PO4)2. Citric acid can also chelate Ca, enhancing Ca-P mineral dissolution (Ström et al. 2005) thus increasing P availability. Thus, the high content of citric acid in Bic exudates at the end of the treatment (33-fold higher than control) could occur in response of both Fe deficiency and low P availability due to high Ca concentrations. In Tric exudates the concentration of citric acid does not show significant difference with –Fe and the amount of malic acid is 3- fold lower: in this last condition the low availability of Fe and P is just a consequence of pH, lacking the effect of CaCO3 on P sequestration. In –Fe conditions the predominance of malate exudation could be attributable to a metabolic state. -Fe plants activate the strategy I mechanisms, increasing ATP and reducing power requirements that cannot be fulfilled through the respiratory chain which is impaired by the lack of Fe. In fact, the impaired mitochondrial electron chain results in a low turnover of NAD+/NADH (Vigani and Zocchi, 2009), thus Fe starved plants have to face the problem of refilling NAD+. The synthesis of citric acid produces NADH+H+, so in a high reductive environment is probably inhibited. In this reductive medium malate could represent a good compromise even if citrate is a better chelating agent. It was widely demonstrated that environmental stresses induce the accumulation of phenolic compounds in plants (Dixon and Pavia, 1995). Phenolics have been reported to have multiple biological effects including antioxidant and chelating activity. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen and free radical scavengers. Furthermore, as phenolics have a metal chelation potential (Rice-Evans et al. 1997) they can contribute through root exudates to facilitate Fe acquisition by plants (Lehman et al. 1987; Dakora and Phillips, 2002; Jin et al. 2006; Cesco et al. 2010). P. judaica growing under directed or induced low Fe availability shows a high accumulation of phenolic compounds both in root tissues and root 89 exudates.No correlation has been found between soluble phenolics concentrations in roots and the progress of treatment. At the end of the treatment however, the concentration of both polar and non-polar phenolics in roots was increased in all stress conditions. The highest amounts of phenolics were recorded in Bic-treated roots, respectively 70% and nearly 4-fold of polar and non-polar phenolics in comparison with their initial contents (tab.3A). This could be explained because a plant growing on a high calcareous condition has to face cumulative stress, due to the low availability of Fe and P as a consequence of the alkaline pH and to the precipitation of P caused by the high content of Ca. Lack of both Fe and P in plants induces high levels of ROS which indicate an oxidative stress (Tewari et al. 2007; Hajiboland, 2007). Many phenolics have antioxidant activities and oxidative stress conditions promote their accumulation. The lower concentration of phenolics in Tric-treated roots with respect to Bic could be due to a lower stress regarding P, as above explained discussing the organic acid content. In –Fe and Tric treatments the concentrations of polar and non-polar phenolics in root tissues increase but do not show significant differences in their values despite the fact that in Tric treatment P. judaica have to cope with the additional problem of P reduced availability respect to –Fe condition. However, as it was pointed out before, the content of citric acid in Tric roots doubles respect to Fe starved roots (tab. 1B) favoring also P availability. Although organic acids have been recognized to play an important role in iron deficiency response, phenolic compounds have been detected earlier in exudates of all treatments. This fact could suggest on one hand that phenolics exudation is a default strategy of P. judaica species, and one the other hand, it could confirm their central role in the environmental stress responses. Phenolic concentrations in root exudates of P. judaica plants subject to direct and induced Fe deficiency are higher than in the control; moreover they significantly increase along the time course. In Bic condition the initial amount of phenolics exuded is lower respect to –Fe and Tric but it increases along the time course reaching concentration equivalent to Tric treatment. In Bic and Tric conditions the content of phenolics at the end of the treatment are similar (4-fold compared to the control) but the 90 highest concentration of phenolics was found in Fe starved condition (10- fold respect to the control). In alkaline conditions the exudation of phenolics most probably achieve the acquisition of Fe as it is present in solution and it is also immobilized in the apoplast (cap. 3, par. 3.3.3) so the plant could reduce the amount of exudates. Comparing the amounts of organic acids (tab. 2) and phenolic compounds (tab. 4) in exudates it can be noted that the latter presents values one order of magnitude higher, this fact is in accordance with many works that reported phenolic compounds as the main components in root exudates (Jin et al. 2007). Jin et al. (2007) have found that when phenolics were removed, the FCR activity and the acidification increased suggesting that they are not involved in the reutilization of apoplastic Fe mediated by phenolics. However, the acidification of the apoplast as well as the rhizosphere could confer structural stability to phenolic compounds. The root apoplastic Fe has been suggested to be an important Fe source for plants as 75% of total Fe in roots is located in the apoplast (Bienfait et al. 1985). The amount of apoplastic Fe in direct and induced Fe deficiency is shown in Table 5. Fe starved roots show a decrease in apoplastic Fe along the time course, reaching a null value at T3. These data are in agreement with findings of Zhang et al. (1991) who found a reduction in the amount of apoplastic Fe as plants were transferred from full nutrient growing solution to Fe starved one. In fact in Fe-deprived conditions the Fe immobilized in the apoplast is the only available source of Fe for the plant. Jin et al. (2007) have demonstrated the involvement of phenolic exudates in the mobilization of apoplastic Fe. The fast depletion of apoplastic Fe in –Fe roots is supported by the high levels of phenolics found in exudates (tab. 4). In alkaline conditions (Bic and Tric) the amount of apoplastic Fe increases due to the high pH which dramatically decreases Fe solubility and induces Fe precipitation. In addition, it has been pointed out that high apoplastic pH depresses FCR activity reducing, as a consequence, the uptake of Fe (Kosegarten et al. 2004). Bicarbonate (Bic) condition shows a higher content of Fe in the apoplast respect to the organic alkaline buffer treatment (Tric). This result could be explained considering the low growth rate exhibited by Bic plants (data not show) causing a lower consumption rate of apoplastic Fe. 91

Two enzymes of the primary metabolism were assayed along the treatment: PEPC and G6PDH (Tab. 6 A and B). Many papers have reported the increased activity of PEPC under Fe starved growing conditions (De Nisi and Zocchi, 2000; Andaluz et al. 2002; López-Millán et al. 2009; Donnini et al. 2010) indicating its key role in Fe-efficient responses. It has been proposed that enhanced activity of PEPC acts in the homeostasis of cytosolic pH and as a driving force to maintain a high rate of glycolysis by consuming PEP, other than producing malate (Zocchi, 2006). The increase in PEPC activity could then maintain an adequate rate of glycolysis required to provide energy supply and hold up reduce based mechanism to acquire Fe. In Fe starved roots a decrease in PK activity was shown (Thimm et al. 2001; Donnini et al. 2010) so PEP seemingly does not follow the TCA pathway directly. It was found a decreasing activity of PEPC along the time course in all treatments. This could be probably due to the narrow localization of the increased PEPC activity to the external layers of cortical cells of root apices where also the H+-ATPase activity is particularly enhanced (Dell’Orto et al. 2002) and the nutrient uptake takes place: as root grows and tissues differentiate, the active portion, in which the PEPC activity is increased, becomes a smaller percentage of the total root biomass, resulting in a dilution effect. Nevertheless, after 7 days under both direct (-Fe) and bicarbonate-induced Fe deficiency the PEPC activity is significantly higher than under normal Fe supply, according to what found in various Fe-deficient species. Regarding the Bic-treated plants, the - presence of HCO3 represents a further alkalinisation of the cytosol and - PEPC activity could decrease the HCO3 concentration, giving a further contribution in controlling the cytosolic pH. On the contrary, in alkaline Tric treated roots, PEPC activity is very low at the end of the time course, suggesting that PEP is probably consumed at an adequate rate mainly via TCA and shikimate pathway to support the mechanism to acquire Fe. In fact in Tric condition Fe can be acquired by the plant and the mitochondrial activity is not impaired. G6PDH is the first enzyme of the oxidative pentose phosphate pathway (OPPP) and is involved in the supply of C5 sugar phosphates formed in the cytosol that can be used as primary substrates for nucleotide synthesis or aromatic aminoacids via the shikimate pathway. G6PDH is also a key 92 enzyme in modulating the redox state of the cell thus preventing oxidative stress (Singh et al. 2012). In fact, NADPH is a strong inhibitor of G6PDH as well as ATP (Singh et al. 2012). In control condition a consistent increase in G6PDH activity was recorded along the time course, while all stress treatments exhibited a reduced activity respect to the control at the end of the experiment (Tab. 6). In particular, the lowest activity was recorded under –Fe condition probably due to a high NADPH/NAD+ ratio, resulting from the impairment of mitochondrial activity and consequent lowered reducing power turnover and energy supply in form of ATP; in fact both the respiratory chain and the TCA cycle, having Fe-containing proteins, are deeply affected under Fe deficiency (Vigani and Zocchi, 2009; Vigani, 2012). The temporary increase in G6PDH activity at T2 could be explained considering that until the second sampling –Fe roots have not depleted the apoplastic source of Fe yet (Tab. 5). Surprisingly also Tric-treated plants, showed very low G6PDH activity along the treatment, without any significant difference with –Fe condition at the final sampling (T4).There is no reason to suppose that NAD(P)H/NAD(P)+ ratio should be high, as mitochondrial activity could not be impaired. A possible inhibition of G6PDH by tricine buffer cannot be excluded. As in Tric treatment phenolic content in roots and exudates are high and some shikimic enzymes are activated (Chap. 4 par. 4.2.4.), we assume that probably the supply of E4P does not come from OPPP in roots but from photosyntates from the shoot. In Bic treatment the activity of G6PDH at the end of the treatment is decreased by 60% respect to the control but it is still higher than under –Fe and Tric treatments, moreover it increases by 60% along the time course with respect to its initial value. The results of the present work show a great flexibility in P. judaica responses to Fe deficiency. Low organic acids and phenolic compounds definitely play a pivotal role in the adaptive strategy performed to cope with Fe deficiency. Both these compounds carry out potentially multiple functions, so P. judaica can modulate their amounts depending on the status and different requirements of the plant. The responses of P. judaica to both alkaline treatments have shown that P. judaica can be defined as a Fe-efficient plant. Furthermore, it has been shown that in - 2+ calcareous treatment the presence of HCO3 and Ca operates in a direct 93

- and indirect way over the Fe bioavailability. As a direct effect, HCO3 - raises the pH decreasing Fe solubility; additionally HCO3 ion buffers the attempt to acidify the apoplast reducing the FCR activity. An indirect effect could be due to the high content of Ca2+ that is preferentially chelate by citrate respect to Fe.

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CHAPTER 4 ______

Low iron availability and phenolic metabolism in a wild plant species (Parietaria judaica L.)* Liliana Tato, Patrizia De Nisi, Silvia Donnini and Graziano Zocchi

*submitted to PPB on dicember 2012 98

ABSTRACT Plant phenolics encompass a wide range of aromatic compounds and functions mainly related to abiotic and biotic environmental responses. In calcareous soils, the presence of bicarbonate and a high pH cause a decrease in iron (Fe) bioavailability leading to crop yield losses both qualitatively and quantitatively. High increases in phenolics were reported in roots and root exudates as a consequence of decreased Fe bioavailability suggesting their role in chelation and reduction of inorganic Fe(III) contributing to the mobilization of Fe oxides in soil and plant apoplast. Shikimate pathway represents the main pathway to provide aromatic precursors for the synthesis of phenylpropanoids and constitutes a link between primary and secondary metabolism. Thus the increased level of phenolics suggests a metabolic shift of carbon skeletons from primary to secondary metabolism. Parietaria judaica, a spontaneous plant well adapted to calcareous environments, demonstrates a high metabolic flexibility in response to Fe starvation. Plants grown under low Fe availability conditions showed a strong accumulation of phenolics in roots as well as an improved secretion of root exudates. P. judaica exhibits enhanced enzymatic activities of the shikimate pathway. Furthermore, the non-oxidative pentose phosphate pathway, through the transketolase activity supplies erythrose-4-phosphate, is strongly activated. These data may indicate a metabolic rearrangement modifying the allocation of carbon skeletons between primary and secondary metabolism and the activation of a non-oxidative way to overcome a mitochondrial impairment. We suggest that high content of phenolics in P. judaica play a crucial role in its adaptive strategy to cope with low Fe availability. Keywords: iron deficiency, metabolic rearrangement, Parietaria judaica, phenolic compounds, shikimate pathway

4.1. Introduction Iron (Fe) is an essential element for life. Due to its redox characteristics Fe plays an important role in cell metabolism. In particular in mitochondrial and photosynthetic electron transport chains it is directly 99

related with energy-yielding processes. Iron is one of the most abundant elements in the Earth’s crust but, in spite of this, in oxygenated environments it is found in ferric forms (Fe(III)), principally oxides and hydroxides that are very stable and scarcely soluble at neutral pH, reaching a concentration far below that required for optimal plant growth [1]. In alkaline soils, mainly calcareous ones, Fe solubility dramatically decreases and it is not readily taken up by the plant, leading to consistent crop yield losses both qualitatively and quantitatively. Accordingly, plants have evolved different strategies to face Fe acquisition. In general Fe acquisition involves two principal strategies in plants: a scavenging strategy and a mining one. Scavenging strategy concern those mechanisms implemented for soil exploration and results in changes in root growth, morphology and architecture, as well as in increased in root hairs density. Mining mechanisms are aimed at mobilizing non bioavailable forms of Fe and include three chemical processes, reduction, acidification and chelation. Obviously these two strategies operate together. Dicots and non Poaceae monocots have evolved a reduction based mining mechanism (Strategy I) localized at the root cell membrane to cope with low Fe availability [2]. Strategy I includes: a Fe specific mechanism of reduction via a transmembrane Fe(III) Chelate Reductase (FC-R), which reduce ferric to ferrous ions that are transported inside the cell by a Fe regulated transporter (IRT); and a non-specific mechanism of protonation by means of the H+-ATPase that extrudes protons acidifying the rhizosphere and consequently increasing Fe solubility [3,4,5]. In addition plants exude a large variety of substances even further acidifying the rhizosphere and acting as organic complexing agents that can contribute to mobilize minerals in soil solution [6]. In many species phenolics have been recognized as an important component of root exudates [7, 8]. Phenolics are a wide group of hydroxylated aromatic compounds found only in microorganisms and plants. They are secondary metabolites that show a huge diversity of structures, from rather simple structures, as phenolic acids, through polyphenols, for instance flavonoids, that comprise several groups, to polymeric compounds such as tannins or lignin. Phenolics play multiple chemical and biological functions in plants mainly related with adaptation to environmental changes [9-12]. They represent a clear example of metabolic plasticity as plants are able to 100

respond to external stresses rapidly inducing their biosynthesis in a reversible way [13]. Countless works reported the increase of phenolic content in plants tissues, roots and root exudates as a response to different biotic and abiotic stresses [14-19]. Among these it has been registered an induction of phenolics secretion by roots under Fe-deficient condition [17, 20, 21]. A multiple role of phenolics as mediators in Fe acquisition has been suggested. Phenolics could operate in three ways: by directly improving Fe solubility mainly due to their reductant and chelating properties; by mediating the reutilization of root apoplastic Fe and by their allelopathic activity influencing rhizospheric microbial communities to produce siderophores and auxin [22, 23]. Hence phenolics can indeed affect nutrient availability [19, 24]. Several studies have underlined that under abiotic stress plants have to face the trade-off in carbon skeletons allocation between growth, maintenance and defence [25-27]. The shikimate pathway is the common route to provide aromatic precursors for further secondary metabolites. It also represents a link between primary and aromatic secondary metabolism. It was estimated that 60% of total plant biomass is composed of molecules that have traversed the shikimate pathway [25]. Successfully coping with a stress conditions such as Fe deficiency presupposes a fine metabolic rearrangement arising from a contrasting physiological requirements. In fact, plants have to manage, on one hand, with an increased demand of ATP and NADH required for Fe acquisition by Strategy I mechanisms concomitant to the impaired mitochondrial transduction of energy and, on the other hand, the carbon skeleton flux shifting towards the production of secondary metabolites. Parietaria judaica (L. 1753) is a wild perennial and sinantropic dicot that from a nutritional point of view behave as an “indifferent” plant. It grows in both acidic and alkaline soils but it usually represents the most widespread flora in highly calcareous and hostile environments such as wall cracks exposed to the sun. In these calcareous environments P. judaica does not show any symptom of chlorosis which is the primary symptom of Fe deficiency. Previous data showed that P. judaica activates all Strategy I mechanisms as a response to Fe deficiency but the degree of 101

activation does not explain the efficiency through which this species adapts to low Fe bioavailability. Significant root morphological modifications have also been observed in Fe-deficient growth conditions [28, 29] High content in phenolics is a characteristic of P. judaica species; nevertheless it has been observed a significant increase of them in roots and exudates when subject to Fe deficiency [29]. In this work we grew P. judaica in different direct or induced Fe-deficient hydroponic conditions: a control full nutrient solution (+Fe), a minus Fe solution (-Fe) deprived of

Fe, an alkaline full nutrient supplemented with NaHCO3 and CaCO3 that brings the pH to 8.3 and mimics a calcareous environment, an alkaline full nutrient solution buffered with Tricine (Tric) adjusted to pH 8.3. Tric treatment was performed in order to discriminate the response caused only by the pH effect from that due to the presence of the bicarbonate ion. We focused our attention on the mechanisms through which P. judaica shifts carbon skeletons from primary to secondary metabolism. We also investigated how primary metabolism rearranges to supply the substrates for shikimate pathway (erythrose 4-phosphate, E4P and phosphoenolpyruvate, PEP) considering that under Fe deficiency there is a greater demand of energy and reducing power as a consequence of the activation of proton extrusion and Fe(III) reduction, and on the other hand an impaired mitochondrial NADH turnover.

4.2. Results 4.2.1. Root modifications Previous works have reported modifications in root morphology in P. judaica grown in Fe deficiency conditions pointing out to the presence of an increased number of secondary roots as well as the onset of clusters of short and thick rootlets [28]. After 7-d treatment we observed results similar to those obtained before. In fact in all treatments we found a shorter but more branched radical system in terms of both adventitious and lateral roots with respect to control. Anyway, it can be noted a different branching pattern in -Fe, Bic and Tric treatment (Fig. 1). –Fe roots develop a lot of very short sketches of lateral roots (Fig.1 B). In particular, we observed a more similar root system in Bic and Tric plants 102

even though shoots show a quite different response. In fact, Bic shoots exhibit a slowdown in growth and more chlorotic leaves respect to Tric (Fig. 1C and 1D).

Figure 1. P. judaica plants obtained after 7d of treatment. A, B, C, D are images of the whole plant in control, Fe starvation (-Fe), bicarbonate (Bic) and alkaline organic buffer (Tric) condition; E, F, G, H are detailed images of the respective root systems. The bar beside the images A, B, C, D indicate the scale.

4.2.2. Phenolic compounds in root and exudates Phenolic concentration was measured in roots and exudates of 7-d-old plants. The content of soluble phenolics found in root extracts of all stress treatments show a higher values increased by 40-50 % respect to +Fe but no significant differences were found between -Fe, Bic and Tric conditions. Total phenolic content obtained through methanolic extraction also show an increase in polar phenolics in comparison with +Fe of nearly 60% for -Fe and Tric treatments while in Bic we found a remarkable increase (nearly 4-fold compared to the control and more than 1-fold respect to the other treatments). Furthermore, it can be 103

noted that in Bic condition the content of polar phenolics is higher than that of soluble ones (Fig. 2 A). Total phenolic content found in field samples collected from alkaline substrates are comparable to those measured in samples grown in hydroponic samples at high bicarbonate content (data not show). In root exudates (Fig. 2 B) a noteworthy increase in phenolics in all stress conditions (5- to 8-fold) with respect to +Fe was detected; while Bic and Tric conditions show comparable phenolic content in exudates, -Fe plants extrude a significant higher amount.

Figure 2. A. Total phenolic content in roots extract with water (w) and methanol (m), B. Total phenolics content in root exudates. Significant differences (P<0.05) are marked with different letters. FW= fresh weight.

4.2.3. Responses of primary metabolism Fe deficiency induces Strategy I mechanisms to facilitate Fe acquisition, among these FC-R and H+-ATPase activities, which require a constant production of energetic substrates. As glucose is the main and most immediate source of energy in plant metabolism, some key enzymatic activities belonging to the glycolytic pathway have been determined. Table 1 shows the results obtained assaying the hexokinase (HK), glyceraldehide 3-phosphate dehydrogenase (GAPDH) and phosphoenolpyruvate carboxylase (PEPC) activities on root soluble extracts. 104

Enzymes +Fe -Fe Bic Tric

HK 67.31 ± 5.20 c 119.76 ± 10.95 a 88.99 ± 8.75 b 81.85 ± 7.69 b

GAPDH 105.60 ± 10.40 d 205.93 ± 15.00 a 143.27 ± 7.80 c 170.63 ± 12.50 b

PEPC 62.54 ± 4.70 b 90.80 ± 8.90 a 83.77 ± 5.70 a 24.61 ± 3.70 c

Table 1. Enzymatic activities of Hk (Hexokinase), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and PEPC (phosphoenolpyruvate carboxylase) measured in dialysed root soluble extracts from +Fe, -Fe, Bic and Tric treated plants. Data are expressed as the means ± SD (n=12). Enzyme activity is expressed as nmol min-1mg-1 prot. Significant differences are labelled with different letters.

HK catalyzes the first step of glycolysis and has been shown to be involved in a complex regulatory mechanism related with shoot and root growth [30]. GAPDH is the first enzyme of the so called energy harvesting glycolytic steps. HK and GAPDH catalysed reactions that generates H+ and could play an important role both in equilibrating the cytosolic pH and in supplying protons for the H+-ATPase [31, 32]. All stress conditions induce an activation of both HK and GAPDH activity confirming the increasing rate of carbohydrate catabolism. -Fe root extracts show activity increase of about 78% for HK and 95% for GAPDH with respect to the control. In bicarbonate supply and tricine buffer conditions HK activity enhancement respect to the control was of 30-20% and no statistical difference was found between them. Regarding GAPDH in Tric treatment, the activity was increase by +62% with respect to the control whereas Bic condition shows a moderate increase (+36%).

- PEPC is responsible for the assimilation of available HCO3 into oxalacetate which is further converted in malate. All Fe deficiency conditions show different PEPC activity. -Fe and Bic extracts exhibit an increase of 45% and 34% in PEPC activity, respectively. On the contrary, Tricine buffered condition shows a marked reduction of PEPC activity (about -60%) with respect to the control. 105

Western blot analyses performed for GAPDH and PEPC are shown in Fig. 3. The results obtained using antibodies against GAPDH reveal more intense bands in all treatments respect to the control. PEPC Western blot results confirmed previous works [29, 33]; all treatments show at least two bands, even more or less evident, corresponding to an apparent molecular mass of 103 kDa and 108 kDa, respectively. A more intense band at 108 kDa was detected -Fe condition with respect to the control, while in Bic treatment it is comparable with control and less intense with respect to -Fe condition. Also in Bic condition the 103 kDa band appears more pronounced than the 108 kDa band. Overall Tric condition show the lower band intensity respect to the other stressed treatments.

Figure 3. Western blot analysis of GAPDH and PEPCase carried out on dialysed root extracts from plants growing for 7 d in control (+Fe), Fe starvation (-Fe), bicarbonate supply (Bic) and alkaline tricine buffered (Tric) condition. The experiment was conducted twice with the same results.

4.2.4. Shikimic pathway enzymes As shikimic acid is a precursor of the biosynthesis of aromatic compounds and consequently phenolics, some enzymatic activities belonging to the shikimate pathway [3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS); shikimate dehydrogenase (SDH); shikimate kinase (SK)] and phenylalanine ammonia-lyase (PAL, the first enzyme of phenylpropanoid pathway) were assayed. Table 2 reports the enzymatic activities analysed.

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Enzymes +Fe -Fe Bic Tric

SKDH 4.38 ± 0.71 c 24.08 ± 1.98 a 14.38 ± 1.34 b 15.26 ± 0.99 b SK 72.15 ± 9.41 c 105.93 ± 8.20 b 170.00 ± 15.76 a 102.23 ± 11.03 b PAL 4.77 ± 0.26 c 8.88 ± 0.39 a 8.80 ± 0.59 a 5.92 ± 0.35 b

Table 2. Enzymatic activities of SKDH, SK and PAL assayed in root extracts of control, -Fe, Bic and Tric treated plants. Data are the means ± SD (n=12). Enzyme activity is expressed as nmol min-1 mg-1 prot for SKDH and SK. For PAL one unit of enzyme activity was defined as the amount of PAL that produced 1 μmol of cinnamic acid within 1 h and was expressed as A290 μmol cinnamic acid/mg protein h-1 for PAL. Differences among data with statistical significance (P < 0.05) are expressed with different letters.

DAHPS is a key enzymatic step in the shikimate pathway since it catalyses the first reaction which condenses phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) giving 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP). Several studies have indicated the existence of DAHPS isoenzymes in plants. Their expression is influenced by many kinds of environmental stimuli, thus modulation of the whole DAHPS activity seems to be the result of differential isoenzyme expression [34, 35]. Unfortunately, DAHPS activity could not be assayed due to the commercial unavailability of the substrate E4P (as mentioned in Materials and Methods). Western blot analysis of DAHPS shows an immunodecoration in correspondence with a polypeptide with the apparent mass of 45 kDa for all treatments (Fig.4). In the absence of Fe a more intense band is present with respect to the control. Surprisingly, Bic condition shows a much weakened band notwithstanding its high content in phenolics. Tric root extracts present an intermediate condition between the control and Bic treatment.

Figure 4. Western blot analysis of DAHPS carried out with root extracts of 7 d plants from control (+Fe), -Fe, Bic and Tric treatments.

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Shikimate dehydrogenase (SKDH) catalyzes the reversible NADPH- dependent reaction of 3-dehydroshikimate to shikimate. In plants SKDH acts as a bifunctional enzyme that catalyzes also the former shikimate step. A significant enhancement of SKDH activity was observed in all treatments respect to the control. In the absence of Fe a 5-fold increase in SKDH activity was shown, while Bic and Tric root extracts exhibit an increase of about 3-fold without significant differences between them. SK is the fifth enzyme of the shikimate pathway and catalyses the specific phosphorylation of the 3-hydroxyl group of shikimic acid using ATP as a co- substrate. SK activity was affected in all treatments showing a significant increase compared with the control. Bicarbonate supply condition shows the highest SK activity (2.5-fold), while –Fe and Tric treatments increase by about 40-50% with respect to the control and do not show significant differences with each other. The ubiquitous higher plant enzyme phenylalanine ammonia-lyase (PAL) is a key biosynthetic catalyst in phenylpropanoid assembly. PAL is the first enzyme in phenylpropanoid pathway and catalyses the non-oxidative deamination of L-phenylalanine to trans-cinnamic acid. As most natural phenolic compounds derived from trans-cinnamic acid PAL is considered to play a pivotal role in channeling carbon flux from primary metabolism to phenolic synthesis. In all stressed growing conditions PAL activity increases. In –Fe and Bic treated plants PAL exhibit a significant enhancement (+86%). A moderate increase of 22% was recorded in the Tric condition. Western immunoblot analysis performed using antibodies raised against PAL is shown in Fig. 5.

Figure 5. Immunological detection of PAL in rootextracts of 7 d-old plants from control (+Fe), -Fe, Bic and Tric treatments.

In purified extracts of roots grown in all treatments two isoforms of apparent molecular mass of 76 and 75 kDa were identified. The 76 kDa polypeptide appears more intense than the 75 kDa one in all lines, moreover Tric treatment extract exhibits a wider marked band. The 75 kDa isoform is more evident in –Fe samples. 108

4.2.5. Oxidative and non-oxidative pentose phosphate pathway activation (OPPP and non-OPPP) Glucose 6 phosphate dehydrogenase (G6PDH) catalyzes the first oxidative reaction of the OPPP converting glucose 6-phosphate in 6- gluconolactone producing NADPH. G6PDH is the rate-limiting step of OPPP. This enzyme is regulated by NADP+ availability. It has been speculated to be an important source of NADPH in non photosynthetic tissues and to be involved in maintaining the redox potential necessary to protect cell against oxidative stress [36]. Transketolase (TK) is considered a key enzyme of the non-oxidative reactions that links OPPP to glycolysis and catalyzes a series of reversible interconversion reactions of sugars. Through TK activity erythrose 4-phosphate and xylulose 5-phosphate can be obtained from glyceraldehyde 3- phosphate (GAP) and fructose 6- phosphate. The enzymatic activities of G6PDH and TK, belonging to OPPP and non- OPPP, respectively and involved in the supply of initial substrates for the shikimate pathway were also analyzed (Tab. 3). Under Fe deficiency conditions (-Fe, +Bic, Tric) the following can be observed with respect to control: a) a lower G6PDH activity; b) a remarkable increase in TK activitiy (–Fe, Bic and Tric activities were enhanced by about 10-, 7- and 3-fold, respectively).

Enzymes +Fe -Fe Bic Tric

G6PDH 56.68 ± 4.30 d 6.23 ± 0.75 a 24.00 ± 2.36 b 7.65 ± 0.81 c TK 13.30 ± 1.24 d 139.88 ± 15.24 a 89.25 ± 8.71 b 35.48 ± 3.12 c

Table 3. OPPP Glucose 6-phosphate dehydrogenase (G6PDH) and non-OPPP Transketolase (TK) enzymatic activities assayed in root extracts dialysed and purified respectively in control, -Fe, Bic and Tric treated plants. Data are the means ± SD (n=12). Enzyme activity is expressed as nmol min-1 mg-1 prot. Statistical differences (P < 0.05) are expressed with different letters.

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Figure 6 shows the immunological localization of a band corresponding to the apparent molecular weight of 74 kDa. This datum is in agreement with what reported in literature [37]. In fact TK is an homotetramer constituted of two dimers of about 74kDa. A very marked band, more intense respect to the control is observed in –Fe treated plants line. In Fe deficiency induced by bicarbonate a more noticeable band in comparison with the control is also detected, while the Tric treatment is comparable with the control.

Figure 6. Western blot analysis of TK carried out on root extracts of 7 d plants from control (+Fe), -Fe, Bic and Tric treatments. (The antibody (tkt3) was raised in rabbits, and the primary antibody dilution was 1:5000;)

4.3. Discussion Plants in natural environment must face continuously with abiotic and biotic stresses. Plant responses to environmental constrains are complexes and involve multiple traits. Anyway, the primary response takes place at metabolic level. Metabolic responses to abiotic stresses concerned interactions and interferences with many molecular pathways. Iron chlorosis is one of the limiting factors in areas of alkaline calcareous soils. Along the evolution, calcicole wild plants have shaped their adaptive responses to these complexes set of environmental stresses. For this reason studying wild plants responses could provide interesting insights useful to increase our knowledge on how plants can overcome these constraints. Increase in phenolic compounds is one of the general stress responses in plants resulting in a shift of carbon flux from primary to secondary metabolism. Moreover, under stress conditions, plants have to cope with the efficient distribution of limited resources between physiological processes and the activation of mechanisms to acquire the depleted nutrients. The metabolic flexibility is therefore the main feature to cope successfully with environmental limitations. A limited availability of nutrients may determine root growth, root proliferation and specific functional responses that depend on genetic makeup as well as the prevailing nutrient status of the plant [38, 39]. 110

P. judaica can grow in highly calcareous environments and displays diversified responses to Fe deficiency that allow it to adapt efficiently. Under Fe deficiency conditions general root morphological modifications have been described in different species. Morphological changes induced by Fe deficiency include swelling of root tips and formation of lateral roots, root hairs, and transfer cells that increase the root surface in contact with the external medium thereby increasing the Fe uptake capability [40-42]. Consistently with previous works [28, 29] it was observed in P. judaica after 7-d of direct or induced Fe deficiency treatments a proliferation of lateral roots that enhance the total absorptive root surface. Albeit the increase of root surface it can be recognized a different branching pattern among the differently treated plants. Lack of Fe induces a high appearance of lateral roots primordia all along the adventitious roots (Fig. 1B and 1F) while Bic and Tric treatments showed shorter root systems with well developed lateral roots distributed in patches (Bic) or uniformly (Tric), respectively (Fig. 1G and 1H). These variances are probably due to the different metabolic status of the plants. In –Fe plants have to face with an absolute lack of Fe that poses severe energetic problems [43]. In fact, on one hand mitochondria have been indicated as the main victim of Fe starvation [44] and on the other hand the biosynthesis of chlorophyll decreases dramatically [45]. The Fe- starved plant has to allocate its restricted energetic resources among searching for available Fe and physiological maintenance of other vital processes. In fact, at root level –Fe plants produce just rootlets that constitute the most active portion of the root system (Fig.1B and 1F). In this way the plant maximizes the response per unit biomass and energy. In Fe alkaline growth conditions Fe bioavailability becomes very low so plants address their efforts to acquire the nutrient increasing the root surface and finally succeeding in retrieve it. However, under bicarbonate supply shoot features denote a slight chlorosis with respect to alkaline tricine buffer condition (Fig.1C and 1D) suggesting a specific stress caused probably by the presence of the bicarbonate ion itself. Phenolic compounds fulfill an important role in the adaptive response to Fe deficiency carrying out important antioxidant and chelating properties [10, 28, 46, 47]. In particular, it was suggested their role in re-mobilizing apoplastic Fe [17]. The results of total phenolics content obtained both in 111

extracts and exudates show a significant increase in all direct or induced Fe starved conditions compared with the control. These data confirm in P. judaica the high increase in these compounds as a response to an external stress. We found the highest content of total phenolics in bicarbonate treated root tissues. Furthermore, in Bic condition the total phenolic content extracted with methanol was higher than that obtained with water (Fig. 2A). This might be attributable to an increased root lignification occurring in these samples (data not show). The release of phenolics through root exudates was greater in -Fe condition while a diminution of about 25% was shown in alkaline conditions (Fig. 2 B). In fact, under Fe starvation plants activate exudation and maintain this response as they do not receive any feedback from Fe uptake. In Bic and Tric conditions Fe is present in the solution in a non available form and precipitates in the apoplast (data not show), so these Fe deposits could be mobilized as the root exudes phenolics. These data suggest that phenolics play a pivotal role in the adaptive strategy of P. judaica to cope with low Fe availability. The activation of reduction processes and the increase in proton extrusion under Fe deficiency requires an increase in ATP, NAD(P)H and H+. Accordingly, an enhancement in the rate of glycolysis could fulfil this demand [32]. Several enzymes of glucose catabolism have been shown to increase their activity [48-52]. In P. judaica we assayed HK and GAPDH which are protogenic enzymes. It was found an increased activity of both enzymes in all treatments, especially in –Fe condition confirming the results found in other species [48]. PEPC catalyzes the fixation of bicarbonate to form oxalacetate/malate. It was pointed out as one of the enzyme that highly increases its activity under Fe deficiency [33, 53, 54]. PEPC plays a central role in Fe deficiency performing a double function: it is involved in the pH stat mechanism in the cytosol and it acts as a driving force for glycolysis [32] consuming PEP that is an inhibitor of phosphofructokinase (PFK). PEPC is responsible for the production of organic acids which are protogenic and can also replenish the TCA cycle. In both direct and induced Fe deficiency treatments the glycolytic enzyme HK, and GAPDH increased significantly their activity, but particularly in -Fe in which they almost double their activity (Table 1). The difference found in glycolytic enzymes and PEPC 112

activities could be looking at the root growth as dry weight (data not shown), while Bic plants have slow down their root growth, Tric roots grow at a comparable rate as the control. This fact could be interpreted as that the energetic substrates resulting from the increased rate of glycolysis are used in Bic treated plants mainly for adaptive processes such as the reduction based mechanism, phenolics and organic acids synthesis, whereas in Tric condition the improved glycolytic activities could be committed both to the increase in biomass and to the adaptive processes. Also in -Fe roots we registered a diminution of growth so in this case the highest rate of carbohydrates degradation through the glycolytic pathway indicates the effort carried out by plants to cope with serious limitations in the energy production due to impaired Fe-dependent mitochondrial activity. In Tric condition PEPC activity is lower than in the control suggesting that probably the consumption of PEP, that is channelled into the shikimate pathway and that forms pyruvate to enter in the TCA - considering that the mitochondrial activity is not impaired - is enough to run glycolysis at an adequate rate (Fig. 7). As primary metabolism is more closely related with growth and development, there is increasing evidence that accumulation of phenolics in plant tissues is a common adaptive response. However, primary metabolism is the source of precursors for secondary metabolism so they are tightly linked. Shikimic pathway is the main way to synthesize aromatic aminoacids, among which phenylalanine (Phe) is a common precursor of numerous phenolics compounds [34]. It has been pointed out that Phe-derived phenolics can constitute up to 30% of organic matter in some species [55]. Actually, around 20% of the total carbon fixed by photosynthesis in land ecosystems is incorporated into lignin, being the second main constituent of plant biomass after cellulose [56]. Furthermore, shikimate pathway represents the bridge through which carbon flux shifts from carbohydrate metabolism to secondary metabolism [57].

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Figure 7. General overview of reactions linking primary and secondary metabolism in P. judaica roots growing for 7 d in control (+Fe), Fe starvation (-Fe), bicarbonate supply (Bic) and alkaline tricine buffered (Tric) condition.

PAL plays a central function at the branch point of phenylpropanoid derivative metabolism and is considered to be one of the key enzymes in the biosynthesis of flavonoids. PAL is also an important enzyme in plant stress response. Its biosynthesis is stimulated by pathogen attack, tissue wounding, UV irradiation, low temperature, or low levels of nitrogen, phosphate, or Fe [18]. Assays conducted on some key enzyme activities of the shikimate pathway and PAL in P. judaica match well with the observed high content and accumulation of phenolics. In almost all cases a high activity of SK, SKDH and PAL corresponds to a high content of phenolics, except for SKDH 114

which only under Bic condition showed a significant increase (Table 2). The Western blot analysis of DAHPS and PAL reveals a different regulation of the enzymatic activity depending on the treatment condition. While - Fe treated plants always show an increase in protein expression, in Bic condition protein expression is usually low despite a high enzymatic activity. In Bic condition it was recorded a significant slowdown in plant growth so it is reasonable to assume that also protein synthesis was inhibited. An interesting question concerns the way to supply the primary substrates for shikimate pathway: E4P and PEP. PEP is obtained by glycolysis the rate of which was observed to be accelerated in all stress conditions. E4P can be supplied through two ways: OPPP and non-OPPP. G6PDH and TK could be seen as the representative enzymes of these two ways, respectively. The first reaction in the oxidative branch of the OPPP catalyzed by G6PDH, is the rate limiting step under physiological conditions. NADPH is a potent competitive inhibitor of the enzyme. Thus, the ratio of NADP+/NADPH regulates the pathway. As the NADP+ level rises, the flux through the pathway increases. The non oxidative branch of the pathway is regulated primarily by substrate availability. Our results provide an interesting scenario. In all treatments an inverse correlation between G6PDH and TK activities was found. These results suggest a distinct change in the way of supplying substrates for the shikimate pathway. This behavior could be explained considering that the respiratory chain is less efficient under Fe deficiency leading, probably, to a lower NAD(P)H turnover [44]. Thus, the shift to a non-OPPP step could be favored, instead of producing more reducing power, through the OPPP. In addition, the non-OPPP way preserves carbon skeletons by avoiding decarboxylation.

In conclusion, Parietaria judaica is a Strategy I plant that implements many other mechanisms that allow it to successfully complete the life cycle in highly calcareous environments. The synthesis of phenolic compounds plays a central role in the adaptive strategy. We could observe that secondary metabolism constitutes the main concern under this stress condition as P. judaica sustains the supply of substrates using non 115

oxidative ways. The data we analysed confirm that under Fe deficiency the metabolic rearrangement takes place by modifying allocation of carbon skeletons between primary and secondary metabolism. We also observe that Parietaria judaica has no problems to acquire Fe in high alkaline environments and we suggest that in a high carbonate environment the availability of bicarbonate itself constitutes the real factor of stress.

4.4. Materials and Methods. 4.4.1. Plant material Cuttings of P. judaica from a mother plant have been obtained and transplanted in an aereated half nutrient solution for 10 days to radicate. Plants were successively transferred to 10 L plastic tanks (40 plants per tank) with four different hydroponic conditions: +Fe (control, full nutrient solution adjusted to pH 6.2), -Fe (full nutrient solution but total absence of Fe brought to pH 6.2), +Bic (full nutrient solution with addition of 0.5 -1 gL CaCO3 and 15 mM NaHCO3 which brought the pH to 8.3), Trc (full nutrient solution buffered with Tricine and adjusted to pH 8.3). Were required pH was adjusted with Na(OH). The treatment buffered with Tricine was introduced to distinguish the low availability of Fe due just to a high pH from the effect of the presence of bicarbonate. Treatments were carried out for 7 days in a grown chamber under 16/18 h light/dark regime with cool-white light 200 mol m-1 s-1, 27/21°C temperature range, 65-75% relative humidity.

The composition of the full solution was as follow: (mM) 0.75 K2SO4, 0.65

MgSO4, 0.5 KH2PO4, 2 Ca(NO3)2, 0.1 FeIII-EDTA, ( M) 10 H3BO3, 1 MnSO4,

0.5 CuSO4, 0.5 ZnSO4, 0.05 (NH4)6Mo7O24.

4.4.2. Collection of root exudates Fifthteen 7-d-old plants for each treatment were transferred to 250 mL distilled water with 10% (v/v) Micropur (Katadyn, Minneapolis, MN) to prevent microorganisms’ activity. Root exudates were collect for a period 116

of 24 h under continuous aeration. The collected exudates were acidified to pH 3.5-4 with HCl and freeze dried. The lyophilized material was resuspended in 3 mL distilled water and filtered through 0.45 m Millipore Millex-HN.

4.4.3. Determination of phenolic content Phenolic content in root tissues was carried out following two different extraction media: 100% distilled water and 100% methanol. Root samples of the different treatments were homogenized in the extraction solution with a volume ratio 1:1 (w/v). The homogenates were centifuged at 10,000g for 15 min. The supernatants were collected. The amount of phenolics in root extracts and exudates was determined spectrophotometrically at 750 nm with the Folin-Ciocalteau reagent using gallic acid as a standard [29]. Seven independent determinations were performed for each treatment.

4.4.4. Soluble enzyme extraction and assay Roots sampled from different treatments were excised, rinsed in distilled water, and homogenate at 2 - 4 °C in one mL of buffer solution per gr of root F.W. with 20% (w/w) PVPP. Buffer solution contained: 50mM Tris-HCl pH 7.5, 10mM MgCl2, 10% glycerol, 1mM 1mM PMSF and 10 mg mL-1 leupeptin. The homogenates were filtered through 4 layers of gauze and centrifuged at 13,000g at 4 °C for 15 min. The supernatants were centrifuged at 100,000g at 4°C for 30 min. The new supernatants were dialysed for 3 h at 4°C against 2 L of the same buffer solution without leupeptine. The dialysed extracts were used for enzymatic assays of Phosphoenolpyruvate carboxylase (PEPC), Glucose-6-phosphate dehydrogenase (G6PD), Hexokinase (HXK) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PEPC (EC.4.1.1.31) activity was determined as in [33]. The reaction was started by adding an aliquot of soluble enzyme extract. G6PD (EC 1.1.1.49) and GAPDH (EC 1.2.1.12) were assayed according to [48]. In G6PDH assay the reaction was started by adding an aliquot of soluble extract, while in GAPDH assay the reaction started with the addition of 1 117

g/mL of 3-phosphoglycerate kinase. HXK (EC 2.7.1.1.) activity was determined as in [58]. The reaction was triggered by adding an aliquot of soluble extract. All the enzymatic activities were assayed by monitoring the absorbance change at 340 nm and were performed at 26°C and in a final volume of 1 mL. Three independent assays were performed for each treatment.

4.4.5. Shikimate pathway enzyme extraction and assay Shikimate dehydrogenase (SKDH, EC 1.1.1.25), shikimate kinase (SHK, EC 2.7.1.71) enzyme extraction and assay were performed as described in [29]. Determination of shikimate pathway enzymes was carried out at 26°C by monitoring the variation in absorbance at 340 nm. Three independent enzymatic assays were performed for each treatment. 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and Phenylalanine ammonia lyase (PAL) extraction and assay DAHPS (EC 4.1.2.15) and PAL (PAL, EC 4.1.1.5) extraction was performed as follows: fresh root samples were homogenized in 100 mM potassium-phosphate buffer (pH 8.0) containing 1.4 mM 2-mercaptoethanol at a ratio of 1:1 (v/w) and 20% (w/w) PVPP was added. The homogenate was centrifuged at 15,000 g for 15 min at 4°C. The supernatant was purified through a Sephadex G-25M column (Pharmacia LKB, Uppsala) previously equilibrated with the same buffer. PAL activity was assayed in purified extracts according to [59]. The absorbance was measured at 290 nm before and after incubation. One unit of enzyme activity was defined as the amount of PAL that produced 1 μmol of cinnamic acid within 1 h and was expressed as μmol cinnamic acid mg-1 prot. h-1. DAHPS activity could not be determined because of the commercial unavailability of substrate erythrose-4-phosphate.

4.4.6. Transketolase enzyme extraction and assay Transketolase (TK, EC 2.2.1.1.) extraction and activity were determined in accordance to [37]. The homogenation was performed in ice, with the addition of 20% (w/w) of PVPP and leupeptine as protease inhibitor. The 118

activity assay was performed by measuring the concentration decrease in NADH at 340 nm. The assay was carried out as in [37] in a final volume of 1 mL.

4.4.7. Protein determination Protein was estimated according to [60] using BSA as a standard.

4.4.8. Polyacrylamide gel electrophoresis and Western blot analysis of enzymes Enzymes were loaded on a discontinuous SDS-PAGE electrophoresis (3.75% stacking gel, 8% separating gel). After SDS-PAGE Western blot analyses were performed as in [33]. Polyclonal antibodies raised against PEPC [33], GAPDH [61], TK [37], DAHPS [62] and PAL [63] were used. Antibodies were diluted as indicated in the literature. Membranes were incubated at room T for 2 h with 1:25,000 diluted secondary antibodies (alkaline phosphatase-conjugated anti-rabbit IgG, Sigma).

4.4.9. Statistical Analysis One way ANOVA analysis was used to test any statistical difference among data. Differences at P <0.05 were considered to be significant. Statistical differences have been expressed by different letters. All Statistical analysis was carried out with SPSS v.15.0.1 (SPSS Inc.)

Acknowledgments We wish to thank Prof. Dorothea Bartels, Dr. Kanna Sato, Prof. M. Royuela and Prof. G. Dobrowolska for providing the antibodies used in this work. This work was done in partial fulfillment for Ph.D. degree of L. Tato. The Authors wish to thank Dr. M. Dell’Orto for her continuous advice during the work and for a critical reading of the manuscript. We are grateful with Dr. Gianpiero Vigani for his valuable graphical assistance.

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CHAPTER 5 ______

Characterization of polyphenols in tissues and exudates of Parietaria judaica undergoing direct and induced iron deficiency growth conditions 126

ABSTRACT Parietaria judaica is a sinantropic perennial weed that displays a high adaptation to live in very disturbed environments such as wall cracks exposed to the sun. In these highly calcareous environments Parietaria does not show any iron (Fe) deficiency symptom. As a Strategy I plant P. judaica activates all Strategy I mechanisms to acquire Fe, anyway it seems that its high content of phenolic compounds in roots and exudates could play a central role in its adaptive response regarding the low Fe bioavailability. In this work a HPLC characterization of phenolic compounds of P. judaica was carried out on samples collected in the field, showing that the most abundant phenolics are some mono- and di-caffeoylquinic derivatives. It was also examined the effect of direct and induced Fe deficiency of plants grown in hydroponics in different conditions on phenolic composition, both quantitatively and qualitatively, in roots and exudates. HPLC analysis revealed that the increase in phenolic compounds induced by Fe starvation, bicarbonate and alkaline buffer treatments in roots had similar HPLC profiles, but showing quantitative differences among the treatments. On the contrary, HPLC profiles of root exudates have shown altered profiles respect to the corresponding root tissues and demonstrate both qualitative and quantitative differences in phenolic compounds composition among root exudates from different growth conditions.

5.1. Introduction Iron (Fe) is an essential micronutrient for major metabolic processes in plants and the energy-yielding electron transfer reactions of respiration and photosynthesis (Guerinot and Ying 1994).Despite its abundance in the Earth’s crust, it is not readily available for plants under oxygenated conditions. The major factor affecting acquisition of Fe by plants is soil pH. In fact, high pH makes Fe, which is mainly present in its oxidized form in well aerated soils, less available leading the concentration of free Fe in soil solution far below that required for optimal growth (Guerinot and Yi 1994). Plants have developed two main strategies to cope with Fe acquisition: Strategy I, in non graminaceous plants, that is based on the reduction of Fe(III) to Fe(II) which is then transported into the cell by a 127

specific transmembrane transporter (IRT1) and Strategy II, in grasses, where particular FeIII chelators (phytosiderophores) are exuded and subsequently transported inside the cell (Römheld 1987, Curie and Briat 2003, Schmidt 2003). In addition to these biochemical mechanisms, Strategy I also comprises physiological, developmental and metabolic mechanisms aimed at adapting the plant to changing levels of available Fe resources. In calcareous environments, where pH ranges from 7.5 to 8.5, Fe bioavailability decreases even more dramatically as it is found in highly insoluble forms. In fact, in calcareous soils crops most frequently display disorders causing the so called lime-induced chlorosis that lead to yield losses both quantitatively and qualitatively (Chen and Barak, 1982, Römheld 1987, Kim and Guerinot 2007, Diaz et al. 2012). Parietaria judaica (L. 1753) is a wild perennial and sinantropic dicot. Under a nutritional point of view P. judaica is an “indifferent” plant as it grows in both acidic and alkaline soils. However P. judaica usually represents the most widespread flora in highly calcareous and hostile environments such as wall cracks exposed to the sun. P. judaica in such environments displays phenotypic changes but does not manifest any chlorotic symptoms. Previous data have shown that P. judaica under Fe deficiency conditions activates Strategy I mechanisms: Fe(III)-chelate reductase and H+-ATPase activities increase as well as the content of low weight organic acids (LOAs) and phenolic compounds in root exudates (Dell’Orto et al. 2003, Donnini et al 2012). Phenolics are a wide group of aromatic compounds found only in microorganisms and plants. Their multiple chemical and biological functions were recognized long ago and involve radical scavenging, UV protectants, insect repellents, antimicrobial activity, pigments, allelophaty and structural functions among others (Hahlbrock and Scheel 1989, Lattanzio et al. 2009). Several classes of phenolics have been categorized on the basis of their basic skeleton: C6 (simple phenols, benzoquinones), C6-C1 (phenolic acids and aldehydes), C6-C2

(acetophenones, phenylacetic acids), C6-C3 (hydroxycinnamic acids, coumarins, phenylpropanes, chromones), C6-C4 (naphthoquinones), C6-C1-

C6 (xanthones), C6-C2-C6 (stilbenes, anthraquinones), C6-C3-C6 (flavonoids, 128

isoflavonoids, neoflavonoids), (C6-C3-C6)2,3 (bi-, triflavonoids, proanthocyanidin dimers, trimers), (C6-C3)2 (lignans, neolignans), (C6-C3)n

(lignins), (C6)n (catechol melanins, phlorotannins), (C6-C3-C6)n (condensed tannins) (Harborne 1980). Phenolic acids are simple phenols that are found usually in a bound form, occurring often in the form ofesters, glycosides and bound complexes. Countless works reported the increase in the amount of phenolic compounds in plants and root exudates as a response to different environmental stresses (Cesco et al. 2010, Treutter2006). Recent studies have suggested a central role of the adjustment of secondary metabolism in the adaptive response to Fe mobilization (Jin et al. 2007). The metal chelating ability of polyphenols is related to the presence of ortho-dihydroxy polyphenols. Notwithstanding many authors have considered the chelating function of some polyphenols as a minor mechanism (Rice-Evans et al 1997). Andjelkovic´ et al. (2006) in an experimental study have demonstrated the Fe biding capacity of dicaffeoylquinic acids (chlorogenic acid in particular). Caffeoylquinic acid (CQA) derivatives are a class of conjugated polyphenolic compounds broadly distributed in plants. The structures ofCQAs are constituted of one or more caffeic acid molecules linked to one or two quinic acid molecules through ester bonds(Fig. 1).

Figure 1. Major caffeoylquinic and dicaffeoylquinic isomers. 5-O caffeoylquinic acid isomer is known as chlorogenic acid. 129

This family of compounds has strong antioxidant properties displaying high cytoprotection activity against ROS, and some of them could display interesting Fe chelating activity. A biomedical research conducted on model cell lines (U937) has suggested that the antioxidantactivity of a series of caﬀeic acid esters and flavonoids is largely mediated by a Fe- chelating mechanism: the catechol ring was identified as playing a pivotal role in this mechanism. In fact, if catechol group is absent,the presence of hydroxy groups did not promote any antioxidant activity (Sestili et al. 2002). Various caffeoylquinic derivatives have been identified in root exudates of different species (Badri and Vivanco 2009) but the studies were principally oriented to evaluate their role as chemical mediators in allelopathy and interactions with soil microbes (Bais et al. 2006). In the present work the phenolic compounds present in roots and leaves of field samples of P. judaica were characterized by HPLC analysis. It was also analyzed the effect of direct and induced Fe deficiency of plants grown in hydroponics in different conditions on phenolic composition, both quantitatively and qualitatively terms, in roots and exudates, with the aim at identifying the responses of secondary metabolism induced by low Fe availability.

5.2. Materials and Methods 5.2.1. Parietaria judaica culture - Field samples: plants were sampled in an urban area of Milan. Samples were collected by breaking the walls and the substrates to obtain whole roots. - Hydroponic culture: cuttings of Parietaria were placed to radicate in aerated half-strength nutrient solution for 10 days. Rooted plants were then transferred to 10 L plastic tanks (40 plants per tank) with four different conditions: +Fe (control, full nutrient solution adjusted to pH 6.2), -Fe (full nutrient solution but total absence of Fe brought to pH -1 6.2), +Bic (full nutrient solution with addition of 0.5 gL CaCO3 and 15 130

mM NaHCO3 which brought the pH to 8.3), Trc (full nutrient solution buffered with Tricine and adjusted to pH 8.3).The pH was corrected with Na(OH) were required. The alkaline treatments were performed in order to distinguish the low availability of Fe due just to a high pH from the effect caused by bicarbonate. The composition of the full solution was prepared as in Dell’Orto et al. (2003). Treatments were carried out for 7 days in a growth chamber under 16/18 h light/dark regime with cool-white light 200 mol m-1 s-1, 27/21°C temperature range, 65-75% relative humidity.

5.2.2. Collection of root exudates Fifteen 7-d-old plants for each treatment were transferred to 250 mL distilled water with 10% (v/v) Micropur (Katadyn, Minneapolis, MN) to prevent microorganisms’ activity. For the collection of exudates, plants were kept during 24 h under continuous aeration in a growth chamber at the same conditions of the previous culture. The collected exudates were acidified to pH 3.5-4.0 with HCl to maintain the structural stability of phenolic compounds and freeze dried. The lyophilized material was resuspended in 3 mL methanol and filtered through 0.45 m Millipore Millex-HN. The filtered solution was then analysed for total phenolic content and HPLC determination.

5.2.3. Isolation and characterization of phenolic compounds in Parietaria judaica For qualitative and quantitative determination of phenolic compounds in plant tissues of Parietaria, roots and shoots (1 g) were first boiled for 3 min in 30 ml MetOH-EtOH (1:1) and then refluxed for 30 min (2x). The solution was then recovered and concentrated under vacuum. As no aqueous fraction was detected the concentrated solution was filtered through 0.45 m Millipore Millex-HN. The filtered solutionswere analyzed for total phenolic content and HPLC determination of phenolic compounds. HPLC analyses were performed with a Hewlett Packard Series 1100 liquid 131

chromatograph equipped with a binary gradient pump G1312A, a G1315A spectrophotometric photodiode array detector was set at 325 nm, and G1316A Column with the thermostat set at 45°C. The Hewlett Packard Chem Station (Rev. A. 06.03) software was used for spectra and data processing. An analytical Phenomenex (Torrance, California, USA) Luna C18 (5 ) column (4.6 x 250 mm) was used throughout this work. The solvent system consisted of (A) MetOH and (B) acetic acid-water (5/95, v/v). The elution profile was as reported by Lattanzio and Van Sumere (1987). The flow rate was 1 ml/min. Samples of 25 l were applied to the column by means of a 25 l loop valve. UV absorption spectra were acquire in the range of 235 to 450 nm. HPLC-MS/MS analyses were performed on a QTrap MS/MS system, from Applied Biosystems (Foster City, CA, USA), equipped with an ESI interface and a 1100 series micro-LC system comprising a binary pump and a microautosampler from Agilent Technologies (Waldbronn, Germany). ESI interface was used in positive ion mode, with the following settings: temperature (TEM) 350 ºC; curtain gas (CUR), nitrogen, 30 psi; nebulizer gas (GS1), air, 10 psi; heater gas (GS2), air, 30 psi; ion spray voltage + 4500 V. Full scan chromatograms were acquired in the mass range 100 – 800 amu, MS/MS chromatograms were acquired at collision energy of 20V. LC conditions were as for HPLC-DAD analyses.

5.2.4. Determination of total phenolic compounds The amount of phenolic compounds in root extracts and exudates was determined spectrophotometrically at 750 nm with the Folin-Ciocalteau reagent using caffeic acid as a standard. The method was adapted according to Cicco et al. (2009). Three replicates of three independent determinations were performed for each treatment (n=9).

5.2.5. Estimation of total ortho-dihydroxy phenolic compounds content (Arnow’s reagent) One mL of extract sample was place in a test-tube and1 mL 0.5 N HClwas added. Tube was well mixed and then1 mL Arnow’s reagent (10 g sodium nitrite and 10 g sodium molybdate made up to 100 ml with distilled 132

water) was added resulting in a yellow colour. After mixing, 1 mL1 N NaOH was added turning to red color. The solution was brought to a final volume of 5 mL with distilled water. Blank was performed without the sample. Absorbance was read at 500 nm. The content of each sample was calculated and expressed as gg-1fresh weight. Chlorogenic acid was used for the standard curve prepared in a range of 0-0.15 mg/mL. Three replicates of three independent determinations were performed for each treatment (n=9).

5.3. Results and Discussion 5.3.1. Phenolic characterization of Parietaria judaica collected from the field

A 2 5

1 3

Peak 1 Peak 2 Peak 3 Peak 5 Figure 1. A. HPLC-DAD chromatogram of methanolic extracts of root samples of Parietaria judaicacollected from the field, B. UV spectra of the main peaks. 133

The characterization of field samples of roots and leaves of P. judaica were analyzed by HPLC-DAD and MS/MS. Figure 1A shows the HPLC profile of P. judaica roots collected from the field. Six peaks were identified in roots according to retention times and UV absorption spectra. The main peaks were also analysed by MS/MS (Tab. 1). Peaks 2 and 5 exhibit UV spectra with a maximum at 325-328 nm and a shoulder at 295-310 nm which is consistent with caffeoylquinic acids spectrum (Fig. 1B). Peak 2 showed a parent molecular mass m/z at 355 and characteristic MS2 ion at m/z 181.1 indicative of the presence of caffeic acid moiety (Tab. 1). Peak 5 showed a deprotonated molecular ion at m/z 517 and a fragmentation at m/z 355 consistent with the chlorogenic moiety and m/z 181.1 diagnostic of the presence of caffeic acid. Peaks 2 and 5 were identified as 5-O-caffeoylchinic acid (chlorogenic acid) and 3,5-O-dicaffeoylchinic acid, respectively. Peaks 2 and 5 represent the most abundant constituents in roots and account for 40% and 48% of the total content of phenolics respectively.

2 Peak Rt max MS MS Phenolic cc Identification (min) (nm) (m/z) (m/z) ( g g-1 FW) Field root 7493.60 1 5.139 325 - - 85,16 3-O-Caffeoylquinic ac. 2 8.051 325 355 181.1, 163.1, 145.1, 3006.27 5-O-Caffeoylquinic ac. 135.1 (Chlorogenic acid) 3 10.789 310 - - 118.81 1,3-O-Dicaffeoylquinic ac. (?) 4 20.139 325 - - 314.83 4,5-O-Dicaffeoylquinic ac. 5 20.837 330 517 355.2, 337.2, 319.2 3571.35 3,5-O-Dicaffeoylquinic ac. 6 25.605 325 - - 286.89 3,4-O-Dicaffeoylquinic ac. Field leaves 5734.74 1 5.321 320 - - 373.63 3-O-Caffeoylquinic ac. 2 7.331 328 - - 307.00 3 8.068 325 - - 1335.42 Chlorogenic ac. 4 12.448 330 - - 3056.00 Caffeic ac. derivative 5 17.423 315 - - 358.43 Coumaric ac. derivate 6 22.656 330 - - 345.35 Quercetine 3 glucoside 7 25.609 313 - - 86.04 Coumaric acid derivative

Table 1. Retention time (Rt), wavelengths of maximum absorption in visible region ( max), mass spectral data, total concentration and concentration of main components identified in field samples of roots and leaves of Parietaria judaica. Data acquisition parameters were performed as indicated in paragraph 5.2.3.

134

Peak 3 presents UV spectra with a maximum at 310 nm and very slight shoulder. Considering the UV spectra and retention time the compound could be identified as a coumaric acid derivative but present in a very low content (1.5% of total phenolic content).Other minor compounds identified are: 3-O-Caffeoylchinic acid known also as isochlorogenic acid (peak 1), 4,5-O-Dicaffeoylchinic acid (peak 4) and 3,4-O-Dicaffeoylchinic acid (peak 6) that together represent less than 10% of total phenolic compounds. The main constituents of field roots extracts of P. judaica are represented Chlorogenic acid and 3,5-O-Dicaffeoylchinic acid that constitute together about 87% of the total phenolic compounds present in root extracts (Tab. 1).

3 4

2 5 6

Peak 3 Peak 4 Peak 5 Peak 6

Figure 2. A.HPLC-DAD chromatograms of methanol extracts of field samples of leaves of Parietaria judaica and B.UV spectra of the main peaks.

HPLC profile of methanolic leaf extracts is shown in figure 2A. In leaves 7 135

peaks were identified by considering both retention times and UV spectra. Peak 3 and 4 represent the main components in leaves. Peak 3 was identified as Chlorogenic acid and accounted for a 23% of the total phenolic compounds. Peak 4 is the principal compound and constitutes the 53% of total phenolic compounds in leaves. Considering the retention time (Tab.1) and UV absorption spectra (Fig.2B) peak 4 is not in the sequence of mono or dicaffeoylquinic acids. Instead, it could be a conjugated Caffeic acid derivative. Other hydroxycinnamic compounds were identified: peak 5 which presents UV spectra consistent with a coumaric derivative, peak 1 probably corresponding to 3-O-Caffeoylquinic acid and peak 7 which was identified by a standard as 3,4- O-Dicaffeoylquinic acid and is present in a very low concentration. All hydroxycinnamic acids in leaves accounted for 93% of the total phenolic compounds. The remaining 7% correspond to a flavonoid (peak 6). Peak 6 presents UV absorption spectra with a maximum at 270 and 370 that correspond to quercetine. The retention time suggests that peak 6 could be a quercetine-3-glucoside. In leaves as in roots phenolic compounds composition is represented mainly by phenolic acids that belong to the hydroxycinnamic class. Even though, the HPLC profiles of leaves and roots are not similar. In leaves the highest concentration is attributable to a caffeic acid conjugate not yet identified whereas in roots the major component corresponds to 3,5-O-Dicaffeoylquinic acid. Chlorogenic acid constitutes an important component in both leaves and roots. In quantitative terms a higher amount of total phenolic compounds of about 23% was found in roots respect to leaves.

5.3.2. Phenolic compounds in roots from different hydroponic treatments (+Fe, -Fe, Bic and Tric) HPLC qualitative and quantitative analyses were carried out in root extracts and root exudates collected from plants undergone direct or induced Fe deficiency growth conditions.

136

A 2 6

3 5 1 4 7

Continued Figure 3. A. Comparison of HPLC-DAD chromatograms of root samples of Parietaria judaica subject to direct or induced Fe deficiency conditions. Control condition is represented by the black trace=control; red trace=Fe starvation (-Fe); blue trace=bicarbonate treatment (Bic) and green trace=alkaline organic buffer condition (Tric). 137

continued B

Peak a Peak 3 Peak 4 Peak 7

Figure 3.B. UV absorption spectra of some peaks of HPLC chromatogram roots grown in different hydroponic conditions.

Many studies have characterized root exudates in different species (Badri and Vivanco 2009) but no work has been done regarding the profile differences in phenolic composition in both roots and exudates as a consequence of low Fe availability. In Figure 3 a comparison of HPLC profiles of root tissues of P. judaica grown in control conditions (+Fe), Fe starvation (-Fe), calcium bicarbonate condition (Bic) and a high alkaline organic buffer condition (Tric) is shown. Root extracts of different treatments have shown very similar HPLC profiles among each other. By comparing retention times and UV spectra 8 peaks (1, 2, 3, a, 4, 5, 6 and 7) were detected. The most abundant phenolic compounds identified in roots grown in different hydroponic treatments belong to the family of hydroxycinnamic acids derivatives, mainly mono and dicaffeoylquinic acids. In control roots (+Fe)(Fig. 3) no significant difference were found in the HPLC profiles respect to field roots (fig. 1A). All peaks correspond in the different treatments except for the compound at peak 4 in +Fe condition. Peaks 2 and 6 were identified as Chlorogenic acid and 3,5-O-Dicaffeoylquinic acid.Peak 1 is present in all root extracts in low quantities and was identified as 3-O-Caffeoylquinic acid. Chlorogenic acid represents the most abundant compound found in roots in all treatments accounting for 57% to 69% of the respective total phenolic content (tab. 2). Minor peaks were 138

identified as dicaffeoylquinic acids isomers: peak 3 corresponds to 1,3-O-Dicaffeoylquinic acid, peak 5 corresponds to 4,5-O-Dicaffeoylquinic acid and peak 7 to 3,4-O-Dicaffeoylquinic acid. Peak “a” is present only in Bic and –Fe condition. UV-absorption spectra (fig. 3B) and retention time (tab. 2) indicate that it could be identified as a caffeic derivative. In Bic peak “a” exhibit a higher amount but it constitute only a 4% of its total phenolic compounds (tab. 2). Peak 4 is present in control and –Fe roots it correspond to a low amount and considering the UV-absorption spectra could be identified as a caffeic derivative. In roots under direct or induced Fe deficiency the total phenolic content increases. Higher increase was detected in roots subject to bicarbonate condition (Bic) with an increase of 73% respect to the control. The main components of the phenolic composition are Chlorogenic (peak 2) and 3,5-O-Dicaffeoylquinic acid (peak 6) together accounting for about 80-90% of the total phenolic contents in all conditions. However the ratio between the main compounds (Chlorogenic/3,5-O-Diccafeoylquinic acid) is slightly higher in -Fe, Bic and Tric conditions respect to the control, revealing a relative augmentation in chlorogenic acid. In comparison with the phenolic components of the control (+Fe), the increase in total phenolic content in stress treatments is principally due to the increase of Chlorogenic acid (peak 2) and the supposed 1,3-O-Dicaffeoylquinic acid (peak 3). Several studies have reported the increase in Chlorogenic acid in response to different abiotic stresses such as wounding(Cantos et al. 2001), increased UV-B irradiation and insect attacks (Izaguirre et al. 2007). Moreover, Fe deﬁciency, as an abiotic stress for plants, was shown to affect the expression and the activity of certain peroxidase isoenzymes and to induce secondary oxidative stress in dicotyledonous species (Ranieri et al., 2001). The increase in Chlorogenic acid could be explained because of its role as antioxidant and free radical scavenger (Niggeweg et al. 2004). Table 3 summarizes the total phenolic content in roots estimated through the Arnow reagent which determines the content in ortho-dihydroxy phenolic compounds.

139

Peak Treatment Identification +Fe -Fe Bic Tric 1 7.15 7.65 10.18 10.36 3-O-Caffeoylquinic ac. 2 487.43 592.23 1028.91 659.78 Chlorogenic ac. 1,3-O-Dicaffeoylquinic ac. or 3 20.16 58.92 47.29 26.89 Caffeic ac.

a - 31.64 72.13 - Caffeic derivative 4 10.71 10.57 - 5.30 probably Caffeic derivative 5 21.20 11.35 10.11 12.66 4,5-O-Dicaffeoylquinic ac. 6 285.81 206.21 272.15 254.08 3,5-O-Dicaffeoylchinic ac. 7 21.30 206.21 19.19 20.59 3,4-O-Dicaffeoylquinic ac. Total 853.76 933.49 1477.65 1002.78

Table 2. Quantitative HPLC determination of the concentrations of the main phenolic components express in g g-1FWin roots of Parietaria grown under different treatments (+Fe, - Fe, Bic, Tric).

The results match well with those obtained by HPLC, showing an increase in all treatments. The highest value was obtained in Bic condition with an increase of 87% respect to the control while in –Fe and Trc conditions the augmentation is moderate (+28% and +17%) respectively.

Treatment Total ortho-dihydroxyl phenolics (Arnow reagent) ( g g-1FW) +Fe (control) 1216.87 -Fe 1568.61 Bic 2278.58 Tric 1424.57

Table 3. Total ortho-dihydroxy phenolic content in roots of Parietaria grown in different treatments (+Fe, -Fe, Bic, Trc) estimated with the Arnow reagent.

140

3 7 5 6 1 2

Figure 4. Comparison of HPLC-DAD chromatograms of root exudatesof Parietaria judaica under control conditions (+Fe) black line, Fe starvation (-Fe) red line, bicarbonate treatment (Bic) blue line and alkaline organic buffer condition (Tric) green line.

In Figure 4 the HPLC profiles of root exudates collected from Parietaria judaica under control conditions (+Fe) and subjected to Fe deprivation (-Fe) and induced Fe deficiency conditions (Bic and Trc) are shown. 141

Among all chromatograms 7 main peaks were detected. The results obtain so far by the HPLC analysis show exudate profiles dissimilar to those obtained in root extracts. All peaks found in control root exudates (+Fe) presented UV spectra with maximum absorption at 325 nm and a shoulder between 295-310 nm and retention times range from about 8 to 18 minutes, these characteristic are compatible with hydroxycinnamic acids derivatives. MS/MS analysis could not be performed as exudate samples contain very low concentrations of phenolics. Peak 2 and 3 could be probably Neochlorogenic and Chlorogenic acid, respectively. Peak 4 and 5 are present in control, -Fe and Bic exudates in very little amounts. Peak 5 (Rt 10.724) is also present in root tissues of all treatments (fig. 3) and could correspond to 1,3-O-Dicaffeoylquinic acid. Peak 6 and 7 are present in all exudates but constitute a major component in Bic ones. The amount of ortho hydroxyl substituted phenolic compounds is presented in table 4. The highest content of ortho hydroxyl phenolics was found in exudates from Fe-deprived roots (3-fold respect to the control) whereas Bic and Tric exudates show similar values compared to the control. This result confirms the higher exudation of phenolics by –Fe roots respect to the other treatments (cfr. Chapter 3, par. 3.3.2.). Bic and Tric amounts, on the contrary, are lower respect to those obtained by the Folin Ciocalteu method.

Treatment Total ortho-dihydroxyl phenolics in root exudates (Arnow reagent) ( g g-1 FW) +Fe (control) 49 -Fe 161 Bic 52 Tric 38

Table 4. Amount of ortho-dihydroxy phenolics in root exudates.

142

Fe deficiency-induced secretion of phenolics is an important part of a plant’s adaptive strategy as it plays multiple functions in favoring Fe acquisition. The increased secretion of phenolics in Fe deficiency conditions is involved in the mobilization of apoplastic and soil Fe (Jin et al. 2008). Furthermore, exudation of phenolics into the rhizosphere influences selectively some microbial soil species that produce either siderophores or auxins that in turn support Fe acquisition by the plant (Jin et al. 2008). The higher content of phenolics found in the exudates collected from Fe-deprived roots represents an exacerbated response. The complete absence of Fe ― previous work demonstrated that –Fe roots have no apoplastic Fe after 3rd day of treatment (cf. Chapter 3, par.3.3.3.) ― probably avoids a feedback signal to modulate the response keeping activated at high levels the mechanisms to scavenge Fe. In Bic and Tric treatments probably the response is modulated as Fe is present, although in a not readily available form, thus when the plant exudes phenolics Fe can be mobilized through chelation or reduction from the apoplast or the growth medium.

5.4. Conclusions Phenylpropanpids are usually found in plants as derivatives of the three hydroxicinnamic acids: caffeic, p-coumaric and ferulic acid. In Parietaria judaica the main phenolic constituents identified are mono and dicaffeoyl quinic acids derivatives, in particular Chlorogenic acid and 3,5-O-Dicaffeoylquinic acid. Notwithstanding qualitative differences, most of the phenolic compounds identified present a catechol group that has been pointed out as a strong antioxidant and a potential metal chelator (Arts et al. 2003). Chlorogenic acid is known to be involved in responses to different biotic and abiotic stresses in several plant species probably through its ability to interact with ROS (Niggeweg et al. 2004). In roots of P. judaica subject to low Fe availability the accumulation of phenolics was mainly due to the increase of the chlorogenic acid component. HPLC profiles have shown similarities in root phenolic composition while exudates presented different profiles from their producer roots. This could suggest a directed and active mechanism related to phenolic exudation so far. 143

References Andjelkovic´M., Van CampJ., De MeulenaerB., DepaemelaereG., SocaciuC., VerlooM., VerheR., 2006. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups, Food Chemistry, 98:23–31 Arts M.J., Dallinga J.S., Voss H.-P., Haenen G.R., Bast A., 2003.A critical appraisal of the use of the antioxidant capacity (TEAC) assay in defining optimal antioxidant structures, Food Chemistry, 80:409-414 Badri D., VivancoJ.M., 2009. Regulation and function of root exudates, Plant, Cell and Environment, 32:666–681 Bais H.P.,Weir T.L.,Perry L.G.,Gilroy S., Vivanco J.M., 2006. The Role of Root Exudates in Rhizosphere Interactions with Plants and Other Organisms, The Annual Review of Plant Biology, 57:233–66 CantoniO., 2002. Plant-derived phenolic compounds prevent the DNA single-strand breakage and cytotoxicity induced by tert-utylhydroperoxide via an iron-chelating mechanism, Biochemical Journal, 364:121-128 Cantos E., Espin J.C., Tomas-Barberan F. A., 2001. Effect of woundingon phenolic enzymes in six minimally processed lettuce cultivars upon storage, Journal of Agricultural and Food Chemistry, 49:322–330 Cesco S., Neumann G., Tomasi N., Pinton R., Weisskopf L., 2010. Release of plant-borne flavonoids into the rhizosphere and their role in plant nutrition, Plant and Soil, 329(1-2):1-25 Chen Y., Barak P., 1982. Iron Nutrition of Plants in Calcareous Soils, Advances in Agronomy, 35:217–240 Cicco N., Lanorte M.T., Paraggio M., Viggiano M., Lattanzio V., 2009. A reproducible, rapid and inexpensive Folin–Ciocalteu micro-method in determining phenolics of plant methanol extracts, Microchemical Journal, 91:107–110 Curie C., Briat J.-F., 2003. Iron Transport and Signaling in Plants, Annual Review of Plant Biology, 54:183-206

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Dell’Orto M., De Nisi P., Pontiggia A., Zocchi G., 2003. Fe DeﬁciencyResponses in Parietaria diffusa: A Calcicole Plant, Journal of Plant Nutrition, 26(10-11):2057–2068 Díaz I., Delgado A., de Santiago A., del Campillo M.C., Torrent J., 2012. Iron deficiency chlorosis in plants as related to Fe sources in soil, EGU General Assembly 2012, 22-27 April, Vienna - Austria, p.4454 DonniniS., De NisiP., GabottiD., TatoL., ZocchiG., 2012. Adaptive strategies of Parietaria diffusa (M.&K.) to calcareous habitat with limited iron availability, 35(6):1171–1184 Guerinot M.L., Yi Y., 1994. Iron: Nutritious, Noxious, and Not Readily Available, Plant Physiology, 104(3): 815–820 Hahlbrock K., Scheel D., 1989. Physiology and Molecular Biology of Phenylpropanoid Metabolism, Annual Review of Plant Physiology and Plant Molecular Biology, 40:347-369 Harborne J.B., 1980. Plant phenolics, In: Bell E.A., Charlwood B.V. (eds.), Enciclopedia of Plant Physiology, New Series, Vol. 8, Secondary Plant Products, Springer-Verlag, Berlin, Germany, pp. 329-402 Imsande J., 1998.Iron, sulfur, and chlorophyll deficiencies: a need for an integrative approach in plant physiology, Physiologia Plantarum, 103:139-144 Izaguirre M.M., Mazza C.A., Svatos A., Baldwin I.T., Ballare C.L., 2007.Solar ultraviolet-B radiation and insect herbivory trigger partially overlapping phenolic responses in Nicotiana attenuate and Nicotiana longiﬂora, Annals of Botany, 99:103–109 Jin C.W., You G.Y., Zheng S.J., 2008.The iron deficiency-induced phenolics secretion plays multiple important roles in plant iron acquisition underground, Plant Signaling & Behavior, 3(1):60-61 Kim S.A., Guerinot M.L., 2007. Mining iron: Iron uptake and transport in plants,FEBS letters, 581(12):2273–2280 Lattanzio V., Kroon P. A., Quideau S., Treutter D., 2009. Plant Phenolics – Secondary Metabolites with Diverse Functions, in: Daayf F., Lattanzio V. (eds), Recent Advances in Polyphenol Research, Volume 1, pp.1-35. 145

Marschner H., 1995. Mineral nutrition of plants, Academic. 2nded - San Diego, CA (USA). Niggeweg R., Michael A. J., Martin C., 2004.Engineering plants with increased levels of the antioxidant chlorogenic acid, Nature Biotechnology, 22:746–754 Ranieri A., Castagna A., Baldan B., Soldatini G.F., 2001.Iron deﬁciency differently affects peroxidase isoforms in sunﬂower, Journal of experimental Botany, 52(354):25–35 Rice-Evans C.A., Miller, N.J., Paganga, G., 1997. Antioxidant properties of phenolic compounds, Trends in Plant Science, 2:152–159. Römheld V., 1987. Different strategies for iron acquisition in higher plants, Physiologia Plantarum, 70(2):231–234 Schmidt W., 2003. Iron solutions: acquisition strategies and signaling pathways in plants, Trends in Plant Science, 8(4):188-193 Sestili P., DiamantiniG., BediniA., CeroniL., Tommasini I., TarziaG., Treutter D., 2006. Significance of flavonoids in plant resistance: a review, Environmental Chemistry Letters, 4(3):147-157 VermerrisW., NicholsonR., 2006. Phenolic Compound Biochemistry, Springer Netherlands Whipps J. M., 1990. Carbon economy, in: Lynch J. M. (ed.)The rhizosphere, John Wiley and Sons, Chichester – West Sussex (UK), pp. 59- 97

Chapter 6 ______

Morphological and architectural modifications of the root system of Parietaria judaica in response to different Fe deficiency conditions*

* this work was carried out in collaboration with Prof. Maria Rosa Abenavoli and Dr. Agostino Sorgonà – Dipartimento di Agraria – Università Mediterranea di Reggio Calabria 146

ABSTRACT Parietaria judaica is a calcicole wild plant that exhibits a high adaptation to Fe low bioavailability environments. As a Strategy I plant Fe-deficiency induces in P. judaica roots all the biochemical mechanisms aimed at favouring Fe availability and acquisition. The high efficiency by which P. judaica deals with Fe-deficiency was linked to the diversified and integrated responses it can perform. Among the most critical environmental factors, nutrient availability can profoundly shape root architecture. Root morphological plasticity could play an important role in P. judaica adaptability. In this study we have analyzed how changes in growth parameters root mass ratio (RMR), root finesness (RF) and root density tissue (RDT) of the root of P. judaica have been able to determine the RLR pattern in response to different experimental treatments. The changes in root morphology and architecture of P. judaica subject to Fe-deficiency direct or induced by the presence of bicarbonate, were examined, focusing on the relationship between the shape and the function of the root.

6.1. Introduction Root architectural plasticity, the ability to exhibit morphological and physiological responses to a changing environmental factor, may play an important role in plant adaptation to heterogeneous environments (Sultan, 2000; Pigliucci, 2001). Root branching is essential to increase the surface area of the root system, enabling the plant to tap more distant reserves of water and nutrients and improve soil anchorage. Plasticity may increase overall resource acquisition efficiency by allowing a plant to respond dynamically to temporally and spatially limited resources (Hutchings and de Kroon, 1994; Huang and Eissenstat, 2000). Consequently, root architectures among and between species vary greatly in form and subsequently in function and appear to be correlated with adaptation and productivity in specific environments (Fitter et al. 1991). 147

Within soil, nutrients are distributed irregularly both in space and time Caldwell, 1994) and, consequently, plants have developed mechanisms to modify their root morphology and physiology in response to availability of nutrients. Such plant plasticity is strongly associated with many functional root traits including root elongation (Cahill and Casper, 2000) and its spatial influence (Casper et al., 2003), root architecture, and uptake capacities among others. Studies on plasticity of functional root traits involved in nutrient acquisition have focused mainly on root length, a morphological parameter that best describes the ability of the root to explore the soil (Ryser, 1998). Root proliferation in response to nutrient deficiency has been the most investigated functional root trait. Parietaria judaica is a wild species that grows in calcareous soils characterized by high concentrations of bicarbonate and a alkaline pH. Despite the bioavailability of nutrients, of Fe in particular, is a limiting factor for plant spreading in such soils, P. judaica is well adapted to these environments and does not show typical symptoms of chlorosis due to Fe- deficiency. Previous work has suggested a physiological strategy that allows P. judaica to adapt to Fe low availability integrating the typical biochemical responses of a Strategy I plant with root exudation of organic compounds, changes in root morphology and a rearrangement of its metabolism (Chap. 4). Reports of morphological changes of the root system in response to varying Fe availabilities mostly refer back to earlier work and describe exclusively adaptations to Fe deficiency (Römheld and Marschner, 1981; Landsberg, 1986). Fe deficiency also induces the ectopic formation of root hairs (Schmidt et al., 2000) by modulating the length, position, and abundance of root hairs (Perry et al., 2007). Moreover, low Fe availability frequently leads to the formation of branched root hairs (Müller and Schmidt, 2004). In this work changes in the root morphology and architecture of P. judaica subject to direct or induced Fe-deficiency conditions were analysed, focusing on the relationship between the shape and the function of the root.

148

6.2. Materials and Methods 6.2.1. Plant material and growth conditions Plant material and growth conditions were performed as reported in Chapter 2 par. 2.2.1.2. All treatments were carried out in hydroponics.

6.2.2. Root morphology and biomass allocation After 7d of treatment 5 plants of each treatment were collected randomly. Shoot and root were separated. Shoot dry weight (WS, g) was measured after drying in oven at 70°C for 48 h. The roots were divided into two orders: shoot-borne roots and 1st-order lateral. Each root was stained with 0.1% (w/v) toludine blue O for 5 min and then scanned at a resolution of 300 dpi (WinRhizo STD 1600, Instruments Régent Inc.,

Canada) for determining the length of the shoot-borne root (LT, cm), the total length of the 1st-order laterals(LI, cm), the volume of the shoot- 3 3 borne root (VT, cm ), and the total volume of 1st-order laterals (VI, cm ) lateral roots by the WinRhizo Pro v. 4.0 software package (Instruments

Régent Inc.). Then, dry weights of the shoot-borne root (WT, g) and the total dry weight of 1st-order lateral roots (WI, g) were measured after drying in oven at 70°C for 48 h. The total root dry weight (WR, g) was the sum of WT and WI, and the plant dry weight (WP, g) was obtained by the sum of WR and WS. Based on the measurements above, the following parameters were calculated for each root order:

-1 RLR L/WP (cm g ) (1)

-1 RMR = W/WP (g g ) (2) SRL = L/W (cm g-1) (3) F = L/V (cm cm-3) (4) TD = W/V (g cm-3) (5) where RLR is the root length ratio, which expresses the root order’s potential for the acquisition of below-ground resources; the RMR is the root mass ratio, which indicates the relative biomass allocated to the root; the SRL, F and TD are the specific root length, fineness and tissue density, respectively, which represent the structural root parameters. L, 149

W and V indicated the length, the dry weight and the volume of each root order, respectively. As reported by Ryser and Lambers (1995), the following relationships can be obtained among the above parameters: RLR = RMR x SRL (6) SRL = F/TD (7)

The number of the 1st-order laterals (NI) was counted directly from the images of each root order. The average length of the 1st-order laterals [LI

= LI/NI] (cm) was also calculated.

6.2.3. Statistical analysis of data The effects of the different treatments on the root parameters calculated were tested by two-way ANOVA. The data were checked for deviations from normality and homogeneity of variances prior to analysis. Tukey’s post hoc test comparison was applied to test the effect of each treatment at P<0.05. In order to correct for allometric effects (Coleman et al., 1994), the ln-transformed plant dry weight (lnWP) was used as a covariate in analysing the parameters of root morphology and biomass allocation when significant correlations between lnWP and these root parameters were found. The data were checked for deviations from normality and homogeneity of variances prior to analysis and the necessary transformations were carried out. The effects of different treatments on the number and average length of the 1st-order lateral roots were tested by two-way ANOVA. The data were checked for deviations from normality and homogeneity of variances prior to analysis. Statistical analysis was conducted using the Systat v. 8.0 software package (SPSS Inc., Evanston, IL, USA).

150

6.3. Results and Discussion In Figure 1 the effect of the different experimental treatments both on the morphology and the radical architecture of seedlings of P. judaica are shown. The parameters that determine these alterations were quantified using an image analysis system (Fig. 2). The length of the entire root system does not show a significant reduction in response to different experimental treatments (Fig. 2.A.), although it is possible to observe a lower total length in the presence of both Bic(-39%) and Tric (-53%). These results are in agreement with what has been previously observed (Chap. 2) for these treatments. These responses were also observed concerning the total root area (Fig. 2.B.). Bic and Trictreatments showed a decrease in the absolute total root area value respect to the control and –Fe conditions, though there are no statistical differences among mean values of different treatments. Regarding the dry weight of the whole root system no statistically significant differences were found between different experimental treatments (Fig. 2C), although it has been observed an increase (+61%) in dry weight in roots of P. judaica exposed to Fe starvation. Therefore, the length, the surface area, the dry weight of the whole root system of P. judaica seemed not to be affected by direct or induced Fe-deficiency conditions compared to the control. Anyway, these parameters tend to appear lower when the plants are grown in bicarbonate (Bic) or organic alkaline conditions (Tric). A long and extended root system allows the plant a greater acquisition of soil resources; especially those present at low concentrations or scarcely available (Marschner 1995; Tinker and Nye, 2000) such as phosphate (P) (Lambers et al., 2006) and Fe (Maschner, 1995). Therefore, data obtained from the whole root system morphology of P. judaica seem to suggest a better adaptation to direct Fe deficiency respect to that induced by both alkaline conditions (Bic and Tric). Although these results appear to be positively related to the values of the shoot dry weight (data not shown), no relation is found with the content of chlorophyll, which remains lower in –Fe condition respect to the alkaline conditions (Chap. 2 par. 2.3.3.).

151

A B C D

Figure 1. – Root architecture and morphology of Parietaria judaica grown under the following treatments: A. full nutrient solution at pH 6.5 (+Fe, control); B. Fe starved solution at pH 6.5 (-Fe); C. high bicarbonate solution at pH 8.5 (Bic); D. alkaline organic buffer solution at pH 8.5 (Tric). The images at the top and at the center of each panel are, respectively, the entire root system and a single shoot-borne root. The red box indicates the portion of shoot-borne root that has been magnified. At the bottom of the figure there is the enlarged shoot-borne root zone.

These apparently contradictory observations could be explained by studying the functional role of other traits of the root morphology. In particular, such approach provides two additional levels of information. The first one considers some absolute root morphological parameters per unit plant biomass or per unit total root volume, in particular the root length ratio (RLR) and especially how this relative parameter is defined by its components. The second level of information is supplied by the study of the spatial configuration of the root system (root architecture) that considers the types of roots and their distribution in the whole root system.

152

Figura 2. Morphological analysis of the whole root system of P. judaica grown in *Fe, -Fe, Bic, and Tric conditions. F and P correspond to the F-test value and the probability value respectively. No significant differences were found among means (p<0.05; Tukey test). n=3.

Many authors have considered the root length ratio (RLR) as a morphological trait associated with the ability of the plant to acquire the nutrients from the soil (Boot and Den Dubbelden 1990; Ryser and Lambers, 1995; Ryser 1998; Ryser and Eek 2000; Sorgonà et al. 2007). This approach would avoid the "allometric effects" (Coleman et al., 1994) or the "apparent plasticity" (Weiner, 2004), especially when dealing with the variations of the root length in response to the edaphic conditions of the soil. The results obtained showed that the RLR of P. judaica resulted not significantly affected by the different experimental treatments (Fig. 3A). This suggests that P. judaica while preserving the RLR in response to direct or induced Fe-deficiency maintained a long root system able to explore a greater volume of soil. The correlation between these results 153 and the morphological chlorophyll content (Chap. 2, par. 2.3.3.) supports the hypothesis of the functional role of a long root system in a medium characterized by a low bioavailability of Fe. In addition, the RLR depends on the allocation of biomass (root mass ratio, RMR) and structure (fineness, RF and density of root tissue, RDT) parameters that consitute its major components (Ryser and Lambers 1995). Therefore, plants could increase the length of their root system by increasing the allocation of biomass (RMR) towards the root or by increasing the fineness (RF) and/or by reducing the density of tissue (RDT). In fact, it has been demonstrated that the variation of the RLR under conditions of nutritional deficiencies could be attributable to the variation of its components, RDT and RF, (Ryser and Lambers, 1995, Hill et al., 2006); while, under conditions of low levels of nitrate the RMR becomes a determinant component in the RLR variation (Sorgonà et al. 2007). In Plantago maritima it was observed that the increase in the root fineness/root tissue density ratio and the reduction of RMR are able to cancel the negative effect of salinity on the root length (Rubinigg et al., 2003). The analysis of P. judaica RMR, RF and RDT root parameters was carried out to determine the RLR pattern in response to different experimental treatments. The whole root system showed in –Fe condition a weak non statistically significant increase in RMR (+25%), accompanied by a slight reduction of RF (-15%) and a greater increase in RDT (+34%) which results in a slight reduction of the RLR with respect to the control (Fig. 3). On the contrary, the presence of bicarbonate causes an increase, non statistically significant, of the RMR (+63%) and RF (+77%) which, associated with a significant increase in RDT (+170%), produces a weak RLR increase compared with the control (+5%; Fig. 3).

154

7000 A F = 1.885 ) P = 0.211 -1 6000 5000 4000

3000

2000

Root ratio (cm g length 1000 0 Controllo+Fe –Fe BICBic TRCTric 4000 0.14 0.3 C F = 3.164 D a F = 7.020 ) P = 0.012 B F = 1.702 P = 0.086 -3 0.12

)

P = 0.243 3

) 3000

-1 0.10 ab 0.2 0.08 2000 b 0.06 b 0.04 0.1 1000

Root fineness (cm cm

Root tissue density (g cm 0.02

Root mass(g g ratio

0 0.00 0.0 Controllo Fe BIC TRC Controllo Fe BIC TRC +Fe –Fe Bic Tric +Fe –Fe Bic Tric Controllo+Fe –FeFe BICBic TRCTric

Figure 3. Root lenght ratio and its components (root mass, fineness and tissue density ratio) of the whole root system of P. judaica in control (+Fe), direct Fe deficiency (-Fe), high bicarbonate (Bic) and high alkaline organic buffer (Tric) conditions. F and P correspond to the F-test value and the probability value respectively. Different letters correspond to significant differences among means (p<0.05; Tukey test), n=3.

Finally, Tric treatment has shown a pattern of RMR, RF and RDT variation intermediate between –Fe and Bic conditions (Fig. 3). It could be concluded that the observed responses of the components of the RLR of P. judaica are diversified depending on the experimental conditions imposed. However they are aimed at maintaining unchanged the RLR with respect to the control. In particular, in direct Fe deficiency conditions, P. judaica allocates greater biomass in the root keeping unchanged the values of RF and RDT respect to the control. On the contrary, in high bicarbonate condition (Bic) all components (RMR, RF and RDT) registered an increase, more consistent in terms of tissue density (RDT). The intra-root analysis could provide a first indication of the root architecture. This analysis takes into account the number and types of roots that constitute the whole root system and the branching pattern which determines the root architecture. 155

In Figure 1 is shown that P. judaica root system consists essentially of "shoot-borne" roots (any root developed from shoot tissues), according to the nomenclature proposed by Zobel and Waisel (2010), from which lateral root emerge. This structure is due to the cutting system of breeding. In direct Fe deficiency conditions (-Fe), the total length of the "shoot-borne" roots is greater than in the control (+40%), while decreases in the presence of both Bic (-55%) and Tric (-16%) treatment (Fig. 4A). The number of "shoot-borne" roots per root system is greater in -Fe (+52%), while it does not appear modified by the alkaline conditions (Bic and Tric) compared to the control (Fig. 4C). A general decrease of the mean length of the "shoot-borne" roots was recorded. In Bic condition a higher statistically significant decrese was found (-49%) whilst a non statistically significant decrease was registered in Tric (-26%) and –Fe conditions with respect to the control (Fig. 4E). Regarding lateral roots, in Tric condition a statistically significant reduction of lateral total length was found compared with the control (-82%). A similar trend, although non statistically significant, was also observed in –Fe and Bic conditions (-56% and -70% respectively) (Fig. 4B). The number of lateral roots in P. judaica is modified by the different experimental conditions, although not significant difference was found. In particular, it is slightly reduced in the presence of bicarbonate (-13%), highly reduced in Tric condition (-37%) and slightly increased in Fe- starved condition (+13%) (Fig. 4D). The mean total length of lateral roots is significantly reduced in Tric (-70%) as well as in -Fe (-52%); while in Bic condition it did not show a significant difference with respect to the control (Fig. 4F).

156

300 300 A F = 2.967 B F = 5.669 P = 0.097 P = 0.022

200 200 a

ab 100 100 ab b

Total length of Lateral root (cm)

Total length of root Adventitous (cm) 0 0

) 40

-1 500 C F = 1.166 ) D F = 1.325 35 P = 0.381 -1 P = 0.332 30 400 25 300 20

15 200 10 100 5

Lateral root number (n. plant

Adventitous rootAdventitous number (n. plant 0 0

1.0 E F = 4.260 F F = 7.069 9 P = 0.045 P = 0.012 a 0.8 ab

6 ab 0.6 a a 0.4 b 3 ab Figure 4. Morphological analyses intra-root of P.0.2 judaica in +Fe, -Fe, Bic and Tricb grown conditions. F and P correspond to the F-test value and the probability value respectively.

Different0 letters correspond to significant differencesAverage length of Lateral root (cm) 0.0 among means (p<0.05; Tukey test), n=3.Average length of root Adventitous (cm) +Fe –Fe Bic Tric +Fe –Fe Bic Tric

Figure 5 shows the results regarding the root system branching P. judaica, according to the nomenclature proposed by Dubrovsky and Forde (2012) 157 for the quantitative analysis of the development of lateral roots. In particular, the length of the region defined as "zone of branching", between the formation of the first lateral root up to the point of attachment to the stem, is significantly reduced by the presence of bicarbonate (-55%) and to a lesser extent by the other experimental treatments (-34% in Fe-deficiency, in Tric -31%) (Fig. 5A). In agreement with Dubrovsky and Forde (2012), this region was used to calculate the density of branching, which appears increased significantly in the presence of bicarbonate (+61%) while it seems unchanged by Fe deficiency and by tricine (Fig. 5B ).

A 9 F = 8.544 P = 0.007

6 a

ab ab

b 3

Root branching zone (cm) branchingRoot zone

)

-1 B F = 7.304 P = 0.011 8 a

6 b b 4 b

(n. cmBranching lateral density 0 Controllo+Fe –Fe BicBIC TRC Tric

Figure 5. Root branching analysis of P. judaica in +Fe, -Fe, Bic and Tric grown conditions. F and P correspond to the F-test value and the probability value respectively. Different letters correspond to significant differences among means (p<0.05; Tukey test), n=3. 158

The results obtained regarding the intra-root morphology indicate that different treatments induce different modification in the root branching pattern in P. judaica. Thus, different types of root systems can be defined in comparison with the control: 1. The root system subject to direct Fe deficiency (-Fe) appears to be characterized by a greater total length of the shoot-borne roots attributable to a greater number of this type of roots rather than an increase in their average length (Fig. 1, images at the top of the panel). Furthermore, roots of this type develop a greater number of shorter lateral roots distributed along the main axis, with a similar density to the control, resulting in a reduction of their total length (Fig. 1 images placed at the centre and at the bottom of the panel); 2. The root system in Bic condition has shown a strong reduction of the total length of the shoot-borne roots attributable to a reduction of their average length, as the number of these roots is similar to the control (Fig. 1, images in top panel). The lateral root length shows a lesser reduction with respect to –Fe condition. In fact although the number of lateral roots in Bic condition is lower compared to –Fe condition the average root length is higher (Fig. 1 images placed at the centre and at the bottom of the panel); 3. The root system developed in Tric condition shows similar average branching parameters with respect to the –Fe condition.

The length contribution (%) of the different root types (shoot-borne and lateral roots) to the whole root system length was assessed in order to better understand the effects of the direct and induced Fe deficiency treatments on the root architecture of P. judaica. In agreement with the results of intra-root morphology, Figure 6 shows that the root architecture in -Fe and in Tric are preponderantly characterized by the development of shoot-borne roots, while, in Bic condition a higher development of lateral roots prevailed. 159

140 PRLA A 140 120 PRLL 120

100 100

80 80

60 60

40 40

20 20 (%) length root Lateral %

% Adventitous root length (%) length root Adventitous % 0 0 Controllo +Fe –FeFe BICBic TRCTric

Figure 6. Root architecture of P.judaica in +Fe, -Fe, Bic and Tric grown conditions. F and P correspond to the F-test value and the probability value respectively. Different letters correspond to significant differences among means (p<0.05; Tukey test), n=3.

The diversification of morphological responses of the root system of P. judaica to direct (-Fe) and induced Fe deficiency (Bic and Tric) can be summarized as follows: a) In all stress treatments the morphological component of the root of P. judaica remains relatively unchanged compared to the control; b) The root systems developed by +Fe and –Fe plants are characterized by a preponderance of shoot-borne roots while those grown in the presence of bicarbonate (Bic) showed a proliferation of lateral roots; c) In Bic condition the allocation of biomass (higher RMR) increases. This increment in biomass is distributed in a fine but high density root tissue (RDT); d) The intra-root morphological traits and those related to the components of the root length have shown to be more plastic to the different experimental treatments compared to the morphological parameters of the entire root system. 160

6.4. Conclusion

In the treatments imposed, two nutritional stress conditions can be distinguished on the basis of the pH. On one hand we have a condition of pH 6.2 in which Fe starvation is imposed; on the other hand a high alkalinity condition, induced by bicarbonate or tricine, where a high pH induce a low availability of Fe and other nutrients such as phosphate, zinc, manganese. In Fe-starved conditions, the plant must combine two fundamental needs: the "search of the nutrient" and "the ability to acquire it". The root system developed in direct Fe-deficiency (-Fe) is characterized by long and numerous "shoot-borne" roots that have a greater number of short lateral roots. This could be a good strategy implemented by P.judaica for adapting to a Fe-starved condition. In fact, it was observed that the "shoot-borne" roots function in anchoring but also in the colonization of deep soil layers and show a higher longevity than the finer lateral roots (Canadell et al. 1996, Pregitzer et al. 1997, Gill et al. 2002, Gill and Jackson 2008). In strongly alkaline conditions, such as that induced by bicarbonate, the adaptive response of the plant is focusing especially to make nutrients soluble. In order to facilitate the solubility of nutrients, the plant adopts physiological mechanisms, such as acidification through extrusion of H+ (Zocchi and Cocucci, 1990; Palmgren, 2001; Santi and Schmidt, 2009) and the release of exudates (Welkie, 2000; Curie and Briat, 2003, Jin et al. 2007, López-Millán et al. 2009). From the morphological point of view, however, the proliferation of lateral roots and the increase in tissue density and length of root hairs represent important traits to explore a larger volume of soil and therefore acquire nutrients. In this context, the root system with a high number of lateral roots, as observed in P. judaica in the presence of bicarbonate (Bic), could exert a decisive role in the process of exudation, since it has been observed that lateral roots or their formation points (Jones, 1998; Darwent et al. 2003; López-Bucio et al. 2003) are the most active root types in exuding organic compounds. 161

Moreover, the number of tips may have an important role in the processes of exudation. In fact, they have been observed to be the areas of greatest release of organic compounds (Badri and Vivanco, 2009; Jones et al. 2009 and reference therein). The root system of P. judaica in Bic condition maintains the number of apices of both adventitious and lateral roots similar to that found in the control, suggesting that conservation of such a root system may have a role in adapting to alkaline environments. Another morphological characteristic observed in Bic condition is the high fineness associated with root segments with a high tissue density. A fine root system might better explore the soil at a close distance. This factor is crucial for the less mobile or poorly available nutrients typical of calcareous soils, when the the "depletion zone" of the nutrient is exceed through the root growth. Furthermore, the presence of a high density of tissues in the root system of P. judaica exposed to bicarbonate correlates well with the adaptive success coping with changes in nutrient availability (Wahl et al., 2001; Hill et al. 2006), with the activation of the secondary metabolism (Chap. 2), as such morphological trait was associated with rich sclerophyll and lignin elements (Wahl et al. 2001; Hummel et al. 2007). Finally, the increase in the root tissue density determines a greater ability of the plant to transport water and also a consequent high tensile strength that protects it against mechanical stress (Wahl et al. 2001). This functional role could be important for P. judaica as in calcareous soils the shortage of water and the mechanical strength constitute important factors of stress. More complex seems the response of the root morphology and architecture in Tric condition, in which a greater reduction of all morphological parameters of the whole root system was found (total length, surface area and dry weight) compared to the other treatments. In addition, as previously reported, the pattern of responses of the root parameters RMR, RF and RDT appears intermediate with respect to –Fe and Bic conditions. In Tric-treated roots a greater root fineness was found that is a sign of a good soil exploration The lower number of lateral roots in Tric treatment could suggest an easier Fe acquisition respect to the bicarbonate condition in agreement with previous observations (Chap. 3 and 4). 162

These results show a morphological diversification of the root response of P. judaica to direct or induced Fe deficiency growth conditions in hydroponic, which, however, should also be validated in soil conditions, where the morphological and architecture root response could be more complex.

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Ryser P., Lambers H., 1995. Root and leaf attributes accounting for the performance of fast- and slow-growing grasses at different nutrient supply, Plant and Soil, 170(2):251-265 Santi S., Schmidt W., 2009. Dissecting iron deficiency-induced proton extrusion in Arabidop-sis roots, New Phytologist, 183:1072-84 Schmidt W., J. Tittel, A. Schikora, 2000. Role of hormones in the induction of iron deficiency responses in Arabidopsis roots, Plant Physiology, 122(4):1109-1118 Sorgonà A., Abenavoli M.R., Gringeri P.G., Cacco G., 2007. Comparing Morphological Plasticity of Root Orders in Slow- and Fast-growing Citrus Rootstocks Supplied with Different Nitrate Levels, Annals of Botany, 100:1287–1296 Sultan S.E., 2000. Phenotypic plasticity for plant development, function and life history, Trends in Plant Science, 5(12):537–542 Tinker P.B., Nye P.H., 2000, Solute movement in the rhizosphere, Oxford University Press, New York Wahl S., Ryser P., Edwards P.J., Phenotypic Plasticity of Grass Root Anatomy in Response to Light Intensity and Nutrient Supply, Annals of Botany, 88(6):1071-1078 Weiner J., 2004. Allocation, plasticity and allometry in plants, Perspectives in Plant Ecology, Evolution and Systematics, 6(4):207–215 Welkie G.W., 2000. Taxonomic distribution of dicotyledonous species capable of root excretion of riboflavin under iron deficiency, Journal of Plant Nutrition, 23:1819-1831 Zobel R.W., Waisel Y., 2010. A plant root system architectural taxonomy: A framework for root nomenclature, Plant Biosystems, 144 (2): 507-512 Zocchi G., Cocucci S., 1990. Fe uptake mechanism in Fe-efficient cucumber roots, Plant Physiology, 92:908–911 CHAPTER 7 ______

General Conclusions ― Summary of findings and future research directions ― 166

Plants, due to their sessile nature, must continuously face the changing environmental conditions. In order to cope with constant and diverse challenges, plants must adjust their physiology, growth and development. Plant responses to abiotic and biotic stresses are complex and involve multiple traits. Anyway, the primary response takes place at the metabolic level. Metabolic responses to abiotic stresses concern interactions and interferences involving many molecular pathways. One of the most important issues for plants is to maintain an adequate nutrient supply under fluctuating environmental conditions. In fact, soil nutrients are typically less than 1% by weight readily available for plant uptake. Roots have the primary function of acquiring nutrients thus their phenotypic plasticity to low resource environment is crucial in plant adaptation. To acquire nutrients successfully plant performs two main strategies: scavenging and mining. Mining strategies are aimed at mobilizing nutrients from unavailable forms to available soil solution forms for plant uptake and encompass biochemical and physiological mechanisms implemented by roots such as acidification, reduction or chelation. Scavenging strategies are achieved by morphological and architectural root changes mainly oriented to increase the soil exploration. Scavenging and mining strategies act simultaneously performing an integrated response. Iron (Fe) is an essential micronutrient for plants and animals which fulfills critical catalytic roles in a plethora of proteins in main metabolic pathways such as photosynthesis and respiration. Despite highly abundant in soils, Fe exhibits a very low bioavailability due to the scarce solubility of its oxidized forms. In calcareous soils, as well as in high pH environments, Fe solubility decreases even more dramatically. In fact, in chalky soils Fe deficiency is the cause of a common plant nutritional disorder known as lime-induced chlorosis which leads to a considerable yield loss. Along the evolution, calcicole wild plants have shaped their adaptive responses to this nutritional environmental stress. For this reason studying wild plants responses could provide interesting insights useful to increase our knowledge on how plants can overcome Fe deficiency constraints. Parietaria judaica, a wild sinantropic plant, can grow in highly calcareous environments without showing chlorosis symptoms. P. judaica displays diversified responses to Fe deficiency that allow it to adapt efficiently. In this work the mining and scavenging strategies performed by P. judaica in response to direct and induced Fe deficiency conditions were studied. 167

Physiological, metabolic and morphological root responses were analyzed. It was demonstrated (Chapter 2) that P. judaica, as a Strategy I plant species, responds to direct and induced Fe deficiency by inducing the FC-R and H+-ATPase activities, and increasing the release through exudates of low organic acids, mainly citric acid, and phenolics. A significant increase in organic acids and phenolics was detected also in root tissues of plants grown under Fe deficiency stress conditions. Multiple functions related to Fe deficiency responses have been attributed to these compounds. Citric acid is protogenic and inside the cell could be involved in cytosolic pH control, in feeding the H+-ATPase proton pump and plant Fe transport, while as exudate is a metal chelator, acidifies the rhizosphere. Phenolic compounds are versatile molecules. They are involved in antioxidant and reduction activities and can also act as metal chelators. Thus, inside the cell they probably play a protective role against the ROS formed under stress condition, whereas in the exudates they are involved in Fe mobilization, as source of carbon for soil microorganism and allelopathy. At metabolic level an increase in the PEPC activity was observed only in Fe starved condition while in the presence of bicarbonate the low PEPC activity was attributed to the inhibition exerted by the high concentration of malic acid and of shikimic acid. It was suggested that in the presence of bicarbonate, the PEP produced by glycolysis could in part be channelled into the shikimate pathway and converted into phenolics, which are abundant in this condition. In agreement with the high amounts of phenolics an increase in key enzymes of the shikimate pathway was found, as the first enzyme of the phenylpropanoid pathway (PAL). It was thus suggested that P. judaica responds through a metabolic shift involving particularly the PEP-consuming reactions. Through a time course (Chapter 3) it was analysed the efficiency of P. judaica responses to Fe deficiency. A highly alkaline organic buffered treatment was added to distinguish the effect of the high pH on Fe availability from the responses induced by the presence of high concentrations of bicarbonate. Low organic acids and phenolic compounds definitely play a pivotal role in the adaptive strategy performed by P. judaica to cope with Fe deficiency. However, it was demonstrated that the biosynthesis of phenolic constitutes a preferential way in P. judaica responses, as they were found in greater amounts and were detected first than low organic acids in exudates. Both these compounds carry out potentially multiple functions, so P. judaica can modulate their contents depending on the status and different requirements of the plant. The responses of P. judaica to both alkaline treatments have confirmed that it 168

is a Fe-efficient plant. However, whilst in organic buffered alkaline condition P. judaica has no problems to acquire Fe and displays a non- stressed shoot phenotype, in the presence of high concentrations of bicarbonate the growth rate diminishes significantly and the plant shows a stressed shoot phenotype even if non-chlorotic. These findings suggest that in a highly calcareous environment the availability of bicarbonate itself constitutes a real factor of stress. In calcareous treatment the - 2+ presence of HCO3 and Ca could operate in a direct and indirect way over - Fe bioavailability. As a direct effect, HCO3 raises the pH decreasing Fe - solubility and, additionally, HCO3 ion buffers the attempt to acidify the apoplast reducing as a consequence the FCR activity. It was also suggested that an indirect effect of bicarbonate treatment could be due to the high content of Ca2+ that is preferentially chelated by citrate respect to Fe, making the chelating action of citrate less effective in acquiring Fe.

Increase in phenolic compounds is one of the general stress responses in plants, resulting in a shift of carbon flux from primary to secondary metabolism. Moreover, under stress conditions, plants have to cope with the efficient distribution of limited resources between physiological processes and the activation of mechanisms to acquire the depleted nutrients. The metabolic flexibility is therefore the main feature to successfully cope with environmental limitations. It was then investigated the activity of some key enzymes of the primary metabolism and the way by which P. judaica supplies the primary substrates to sustain the high rate of biosynthesis of phenolic compounds (Chapter 4). The results obtained provide an interesting scenario that suggests a distinct change in the way to supply substrates for the shikimate pathway. The inverse correlation between G6PDH (the first enzyme of OPPP) and TK (an enzyme of the non-OPPP) activities found in Fe starved condition may indicate a metabolic rearrangement by modifying the allocation of carbon skeletons between primary and secondary metabolism. Moreover, the activation of a non-oxidative way could overcome a mitochondrial impairment due to the scarcity of Fe. The shift to a non-OPPP way to obtain erythrose-4-phosphate ― a primary substrate in the shikimate pathway ― could be favored as a consequence of a reduced turnover of NADH, so avoiding an overproduction of reducing power through the OPPP when Fe lacks. In addition, the non-OPPP way preserves carbon skeletons by preventing decarboxylation, thus constituting a carbon saving strategy. The effect of direct and induced Fe deficiency on the composition of 169

phenolic compounds of P. judaica was investigated by HPLC analyses on roots and exudates (Chapter 5). Field samples have shown that the most abundant phenolics are some mono- and di-caffeoylquinic derivatives. The analyses carried out on samples from plants grown in hydroponics in different conditions revealed similar HPLC profiles in roots but showed quantitative differences among the treatments. On the contrary, HPLC profiles of root exudates have shown altered profiles respect to the corresponding root tissues and demonstrate both qualitative and quantitative differences in phenolic compounds composition among root exudates from different growth conditions. The phenolic compounds identified present a catechol group that has been pointed out as a strong antioxidant and a potential metal chelator. Furthermore, chlorogenic acid ― the main component ― is known to be involved in the responses to different biotic and abiotic stresses in several plant species, probably through its ability to interact with ROS acting as a potent antioxidant. It was shown its ability to chelate Fe, thus avoiding the catalysis of Fenton reaction. In roots of P. judaica subject to low Fe availability the accumulation of phenolics was mainly due to the increase of the chlorogenic acid component. Root exudates presented different HPLC profiles from their producer roots so far. This could suggest an addressed and active mechanism related to phenolic exudation.

Under Fe deficiency general root morphological modifications have been described in different species. Morphological changes induced by Fe deficiency include swelling of root tips and formation of lateral roots, root hairs, and transfer cells that increase the root surface in contact with the external medium, thereby increasing the Fe uptake capability. Changes in root morphology were studied in Parietaria judaica under direct or induced Fe deficiency treatments (Chapter 6).Through imaging system analyses it was demonstrated a morphological and architectural diversification of the root system as a response to the direct or induced Fe deficiency conditions. In stress conditions a proliferation of lateral roots that enhance the total absorptive root surface was registered. Albeit the increase of root surface a different branching pattern among the differently treated plants was observed. These differences are probably due to the different metabolic status of the plants. Minus Fe plants produce just rootlets that constitute the most active portion of the root system. In this way the plant maximizes the response per unit biomass and energy. In Fe alkaline growth conditions Fe bioavailability becomes very low, although Fe is still present, so plants address their efforts to acquire Fe increasing the root surface that can succeed in 170

retrieve it. However, under bicarbonate supply root morphology denoted a higher stress condition as the number of lateral roots and tissue density were greater. Moreover shoot features presented a slight chlorosis with respect to alkaline organic buffer condition suggesting a specific stress caused probably by the presence of the bicarbonate ion itself.

In conclusion, the results obtained so far suggest that Parietaria judaica responds to Fe deficiency performing integrated responses which indicate a high plasticity at physiological, biochemical and morphological level. Secondary metabolism plays a pivotal role in Fe acquisition but further research are needed to investigate: - the direct involvement of dicaffeoylquinic acids in antioxidant and chelating activities regarding plant Fe deficiency; - the action of dicaffeoylquinic acids on Fe apoplastic deposits; - the role of dicaffeoylquinic acids as promoters of mutual interactions on soil microorganism; - a metabolomic study to confirm the primary–to-secondary metabolism shifts proposed - the regulation of the main shift (PEP and G6PDH) between primary and secondary metabolism.